[go: up one dir, main page]

WO2001088162A2 - Procedes et vecteurs destines a generer des anticorps dans des especes aviaires et utilisations - Google Patents

Procedes et vecteurs destines a generer des anticorps dans des especes aviaires et utilisations Download PDF

Info

Publication number
WO2001088162A2
WO2001088162A2 PCT/US2001/014817 US0114817W WO0188162A2 WO 2001088162 A2 WO2001088162 A2 WO 2001088162A2 US 0114817 W US0114817 W US 0114817W WO 0188162 A2 WO0188162 A2 WO 0188162A2
Authority
WO
WIPO (PCT)
Prior art keywords
dna
antibodies
cells
sequence
avian
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2001/014817
Other languages
English (en)
Other versions
WO2001088162A3 (fr
Inventor
Lingxun Duan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GenWay Biotech Inc
Original Assignee
GenWay Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/573,442 external-priority patent/US6951742B1/en
Application filed by GenWay Biotech Inc filed Critical GenWay Biotech Inc
Priority to AU2001259631A priority Critical patent/AU2001259631A1/en
Publication of WO2001088162A2 publication Critical patent/WO2001088162A2/fr
Publication of WO2001088162A3 publication Critical patent/WO2001088162A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
    • G16B50/20—Heterogeneous data integration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to processes for producing polyclonal and monoclonal antibodies to an antigen in an avian species, preferably in a chicken, using polynucleotide vaccination.
  • the present invention also relates to processes for determining the proteomics profile of a set of pre-selected DNA sequences isolated from a bio-sample, preferably the proteomics profile of a human cDNA library.
  • the present invention additionally relates to processes for identifying physiologically distinguishable markers associated with a physiologically abnormal bio-sample.
  • the present invention further relates to antibody arrays, integrated databases for identification of genes and proteins, multi-functional gene expression vectors, and methods of producing and using such antibody arrays, integrated databases and multi-functional gene expression vectors.
  • Genetic immunization represents a novel approach to vaccine immune therapeutic development.
  • genetic vaccination has the advantages of relatively short development time, ease of large-scale production, low development, manufacturing and distribution costs, and better safety for the vaccine producers, administers and receipts.
  • Genetic vaccination can be divided into DNA vaccination and mRNA vaccination. Recent studies have revealed the following important features of plasmid DNA immunization (Chattergoon et al, FASEB, 1997, 11:753-763). First, different tissues, based on the delivery method (in particular, the muscle and skin), can be transfected in vivo by plasmid DNA and serve as productive antigen factories. Second, protective cellular and humoral responses can be induced through a variety of delivery methods in some model systems. Third, only small quantities of plasmid DNA are necessary for antigenic stimulation. The success of plasmid DNA immunization in inducing immune responses to several target antigens through several immunization sites and via several unique delivery techniques solidified the concept of DNA vaccines. This technology has since been applied to many disease models including influenza B, hepatitis B virus, malaria, tuberculosis, SIN and HIN type 1 and various cancers (Id.).
  • Polyclonal antibodies have traditionally been produced in mammals such as mice, rabbits, sheep, goats, and pigs.
  • the antibodies are obtained from the serum after an immunization period.
  • This technique is invasive, time consuming and costly, involving restraint of and blood sample collection from the animals.
  • polyclonal antibody production in chickens, especially with the egg yolk as the antibody source is a non- invasive technique.
  • the concentration of immunoglobulin in egg yolk may be similar to that of serum (Altchul et al., Nature Genetics, 1994, 6:119-129).
  • poultry have a lower phylogenetic status than mammals (European Community Directive 86/609 Article 7), and it is therefore desirable to use birds instead of mammals.
  • chickens can often be used as antibody producers (Burke et al., Science, 1987, 236:806-812). Another advantage of chickens as antibody producers is that the chicken antibodies are often useful in assays of the analogue to the antigen present in other species (Bonaldo et al., Genome Research, 1996, 6:791-806; Buckler et al., Proc. Natl. Acad. Sci., 1991, 88:4005- 4009).
  • Fynan et al. Proc. Natl. Acad. Sci, 1993, 90:11478-11482, described DNA vaccination of mice and chicken using purified DNA expressing an influenza hemagglutinin glycoprotein. Fynan et al. found that 67-95% of the test mice and 25-63% of test chickens were protected against a lethal influenza challenge. Protections occurred in both mice and chicken that did not have detectable levels of anti-influenza antibodies before challenge. Before challenge, the DNA vaccination and booster inoculations raised non-detectable or very low level of anti-influenza antibodies in mice. No data concerning antibody response in chicken after the DNA vaccination were described.
  • WO 94/24268 discloses recombinant vectors comprising a recombinant avian adenovirus incorporating, and capable of expression of at least one heterologous nucleotide sequence, which is preferably one which encodes an antigenic determinant of infectious bursal disease virus.
  • WO 94/24268 also discloses methods of production of recombinant vectors, methods of preparation of vaccines based on the vectors, administration strategies and methods of protecting poultry from disease. In the methods disclosed therein, chicks were immunized with functional viruses containing the recombinant avian adenovirus. The antibodies thus generated could only be detected by ELISA.
  • WO 94/24268 does not disclose or teach any steps for recovering the antibodies from the immunized chicks, especially steps for recovering such antibodies from egg yolk or B cells from the immunized chicks.
  • WO 99/02188 discloses egg yolks and egg yolk fractions containing avian antibodies against Clostridium difficile, and methods of providing passive immunization for the prevention or treatment of pseudomembranous colitis or diarrhea by means of enteral administration of anti-Clostridium difficile yolk antibodies harvested from the eggs of hyperimmunized avian hens.
  • the Clostridium difficile antigens used to generate the immune response in hens are somatic antigens, i.e., whole cell antigens, or toxoids of toxins A or B or combinations thereof.
  • WO 99/02188 does not disclose or teach immunization of hens with nucleic acids.
  • the human genome consists of about 60,000-100,000 genes, scattered among 3-4 billion nucleotides of chromosome-based DNA code, the sequencing of which could be completed as early as
  • DNA sequence information provides only a static snapshot of all the possible ways a cell might use its genes. Therefore, this enormous amount of static DNA sequence information needs to be correlated with dynamic information about gene products and their interactions in order to provide meaningful insight for fundamental biological processes and applications of such insight into various fields.
  • proteome was first introduced in July 1995 and was defined as the "total protein complement of a genome" (Wasinger et al., Electrophoresis, 1995, 16:1090-1094).
  • Proteomics aims to supplement gene sequence data with information on what proteins are being made where, in what amounts, and under what conditions (Persidis, Nature Biotechnology, 1998, 16:393-394). It aims to show how protein cascades inside cells change as a result of specific diseases, thereby identifying novel potential drug targets. It then aims to validate particular drug leads against those targets by providing information on how those leads affect the proteome cascades (Persidis, Nature Biotechnology, 1998, 16:100-101). Therefore, in addition to providing answers to fundamental questions about the molecular basis of a cell's state at any point in time, proteomics promises to accelerate novel drug discovery through automated analysis of clinically relevant molecular phenomena.
  • proteomic research is lagging behind.
  • proteomic characteristics including the existence, quantity, cellular location and tissue or developmental expression specificity, of the majority of the proteins putatively encoded by the presently known human DNA sequences have not been characterized.
  • the currently available large-format 2-DE is capable of producing gels containing up to 10,000 distinct protein and peptide spots, over 95% of the spots separated by such 2-DE gel cannot be sequenced because they are beyond the limits of current high-sensitivity Edman sequencing technology (Persidis, Nature Biotechnology, 1998, 16:393-394).
  • Antibodies are conventionally generated by protein or peptide vaccination of mammals such as mice, rabbits, rats or sheep. However, such vaccination is time consuming and costly. Therefore, due to the vast number of the known DNA sequences to be characterized, it is virtually impossible to use the conventional protein or peptide vaccination technology to generate antibodies for large-scale proteomic research.
  • the invention described herein encompasses a process for producing antibodies to an antigen in an avian species, which comprises: 1) delivering to said avian species a DNA sequence encoding said antigen operatively linked to a promoter, said promoter being capable of directing expression of said antigen in said avian species, or a mRNA sequence encoding said antigen, in a amount sufficient to induce detectable production of said antibodies to said antigen; and 2) recovering said antibodies from said avian species.
  • the avian species being vaccinated is a chicken or quail and the antibodies are recovered from egg yolk of the chicken or quail.
  • the present invention also encompasses a process for producing a monoclonal antibody to an antigen in an avian species, which comprises: 1) delivering to said avian species a DNA sequence encoding said antigen operatively linked to a promoter, said promoter being capable of directing expression of said antigen in said avian species, or a mRNA sequence encoding said antigen, in a amount sufficient to induce detectable production of said antibodies to said antigen; 2) removing at least a portion of antibody- producing cells from said avian species; 3) immortalizing said removed antibody-producing cells; 4) propagating said immortalized antibody-producing cells; and 5) harvesting said monoclonal antibody produced by said immortalized antibody-producing cells.
  • the avian species used herein is chicken. More preferably, the chicken antibody-producing cells are immortalized by fusing with cells of a chicken B lymphoblastoid cell line or by oncogene transformation.
  • the present invention additionally encompasses a vector for expressing genes in avian and bacterial cells, which comprises the plasmid depicted in Figures 3A & 3C; and a vector for immortalizing chicken antibody-producing cells, which comprises the plasmid depicted in Figure 12.
  • the present invention further encompasses a process for assessing the proteomics profile of a set of pre-selected DNA sequences isolated from a bio-sample, which comprises: 1) cloning each of said DNA sequences into a dual-expression vector that is capable of expressing said DNA sequences in avian cells, non-avian cells or in vitro expression systems; 2) delivering said DNA sequence in said dual-expression vector formed in step 1), or mRNA or protein encoded by said DNA sequence, or a mixture thereof, to an avian species in an amount sufficient to induce detectable production of antibodies to an antigen encoded by said DNA sequence, and recovering said antibodies from said avian species; 3) delivering said DNA sequence, or mRNA encoded by said DNA sequence, or a mixture thereof, which is delivered to said avian species in step 2), to said non-avian cells, and recovering proteins or peptides encoded by said DNA sequence from said non-avian cells, or expressing and recovering proteins or peptides encoded by said DNA sequence in said in vitro expression
  • the present invention further encompasses an array of antibodies attached on a solid surface.
  • the antibodies used in the array specifically bind substantially to proteins or peptides encoded by a set of pre-selected DNA sequences isolated from a biosample.
  • the present invention further encompasses a method for assessing proteomics profile of a biosample, which method comprises: 1) dividing a plurality of antibodies into an unlabelled portion and a labeled portion; 2) attaching the unlabelled antibodies on a solid surface to form an array of unlabelled antibodies on said solid surface; 3) contacting said array of unlabelled antibodies formed in step 2) with a biosample to retain antigens contained in said biosample that specifically bind to said unlabelled antibodies; 4) detecting said retained antigens by contacting said retained antigens with said labeled antibodies, thereby proteomics profile of said biosample is assessed.
  • the present invention further encompasses an integrated database for identification of genes and proteins, which integrated database comprises a genomic sequence subdatabase, a cDNA sequence subdatabase, a dual expression vector subdatabase which provides information for a plurality of vectors that are capable of directing expression in an avian species and in a non-avian species or an in vitro expression system, a protein sequence subdatabase, an antibody subdatabase and means for linking information in one subdatabase to information in other subdatabases, wherein said genomic DNA sequences, cDNA sequences, dual expression vectors, proteins or peptides and avian antibodies correspond to each other according to the central dogma and antigen-antibody binding specificity.
  • the dual expression vector directs expression in an avian species and in a non-avian species such as a bacterium, a yeast or a mammal.
  • a non-avian species such as a bacterium, a yeast or a mammal.
  • the antibody subdatabase provides information for a plurality of IgY antibodies produced in the avian species.
  • the present invention further encompasses a method for generating an integrated library for identification of genes and proteins, which method comprises: 1) selecting and marking a plurality of DNA sequences from a genomic library; 2) selecting and marking a plurality of cDNA sequences from a cDNA library that correspond to said selected and marked plurality of genomic DNA sequences; 3) cloning said plurality of selected and marked cDNA sequences into a dual expression vector that is capable of directing expression of said plurality of selected and marked cDNA sequences in an avian species and in a non-avian species or an in vitro expression system; 4) producing a plurality of proteins or peptides encoded by said plurality of selected and marked cDNA sequences by delivering and expressing said dual vector containing said plurality of selected and marked cDNA sequences into said non-avian species or said in vitro expression system; and 5) generating antibodies from an avian species using said dual vector formed in step 3) via nucleic acid vaccination or using proteins or peptides formed in step
  • the method further comprises a step of conducting immunoreactions between said antibodies generated in step 5) with said proteins or peptides generated in step 4) to characterize the immunospecificity of said antibodies.
  • the method further comprises a step of conducting immunoreactions between said characterized antibodies with a biosample from which genomic library is isolated to determine the proteomics profile of the selected and marked plurality of genomic DNA sequences.
  • the present invention further encompasses a method for generating an integrated database for identification of genes and proteins, which method comprises: 1) delivering bioimformatic information of the plurality of genomic DNA sequences, the plurality of cDNA sequences, the plurality of dual expression vectors, the plurality of proteins or peptides, and the plurality of avian antibodies obtained using the above-described methods into the corresponding genomic DNA, cDNA, dual expression vector, protein or peptide, and the avian antibody subdatabases; and 2) providing means for connecting the bioimformatic information from one subdatabase to any or all of the other subdatabases.
  • the method further comprises a step of delivering bioimformatic information of the immunospecificity of the avian antibodies obtained using the methods described in the following Section C into the integrated database.
  • the method further comprises a step of delivering bioimformatic information of the proteomics profile of the selected and marked plurality of genomic DNA sequences, which can be obtained according to the methods described in the following Section C, into the integrated database.
  • Figure 1 depicts a method for selecting DNA clone of interest.
  • FIG. 2 depicts a diagram of the antibody assisted method for identification of gene and protein (AMIGAP).
  • Figure 3 A depicts restriction map of pS&DV
  • Figure 3B depicts the construction pS&DV
  • Figure 3C depicts restriction map of pS&DV-S
  • Figure 3D depicts the construction pS&DV-S.
  • Figure 4 illustrates potential immune response elicited by DNA vaccination.
  • Figure 5 depicts ELISA titering of antibody produced in chicken by DNA vaccination with three antigens encoded by pCMV-HBx, pCl-HBN-pol and pZeoSN2- hCD34.
  • Figure 6 depicts a restriction map of HbxAg antigen specific expression vector pCMV-HBx.
  • Figure 7 depicts a restriction map of Hepatitis B virus Polymorantz antigen specific expression vector pCI-HBV-pol.
  • Figure 8 depicts a restriction map of human CD34 antigen specific expression vector pZeoSV2-hCD34.
  • Figure 9 depicts binding affinity of IgY produced by DNA vaccination with HBxAg.
  • Figure 10 depicts SDS-PAGE analysis of IgY purified from egg yolks.
  • Figure 11 depicts Western Blot analysis of anti-HBxAg IgY produced by DNA vaccination.
  • Figure 12 depicts restriction map of plmmo vector which can be used for immortalizing chicken B cells.
  • Figure 13 illustrates a prototype of antibody-chip and its operating procedures.
  • Figure 14 illustrates the concept of the Integrated Databases for Identification of Genes and Proteins (IDIGAP). Shown in the figure are five databases, i.e., genomic, cDNA, expression vector, recombinant protein, and antibody databases, with their relationship and linkage structure.
  • the network formed by the databases has the items corresponding to each other in each individual database so that the network can be used for determination of quantitative or qualitative changes of a given protein in tissues or cells;
  • the protein identified is considered as a target protein which can be used for further identification of its location, function, or relation to certain biological or physiological, or pathological status.
  • the identity of the target protein can be used to pick out the corresponding gene that has the same identification label in the genomic database.
  • Figure 15 illustrates exemplary use of the IDIGAP. Shown in the figure is the process and specific steps for application of the IDIGAP technology. Specifically, a process utilizing IDIGAP for identifying a disease-related protein and gene is depicted.
  • Figure 16 illustrates an exemplary multi-functional gene expression vector (pMFGEV). Modes of Carrying Out the Invention
  • Antibody production normally requires the use of laboratory animals (mostly rabbits, but also mice, rats and guinea pigs) or larger mammals, such as horses, sheep, and goats.
  • the procedure involves two steps, each of which not only causes distress to the animals involved but also is very expensive and labor intensive: a) the immunization itself; and b) bleeding, which is a prerequisite for antibody preparation.
  • avian species such as chickens or quail for antibody production, as opposed to mammals, represents both a refinement and a reduction in animal use. It is a refinement in that the second painful step, i.e., the collection of blood, can be replaced by antibody extraction from egg yolk. It entails a reduction in the number of animals used because chickens produce larger amounts of antibodies than laboratory rodents, h fact, it has been known that the immunization of a chicken induces the production of similar concentrations of specific antibodies in both egg yolk and serum.
  • the use of antibodies produced from an avian species, e.g., chicken can be advantageous in certain circumstances.
  • the immune response in an antibody- producing animal tends to increase as its phylogenetic difference with the animal used as the antigen source increases.
  • chicken antibodies recognize more epitopes on a mammalian protein than the corresponding rabbit antibody does, making it advantageous to use IgY in immunological assays of mammalian proteins. This is especially true when the antigen is a highly conserved protein, such as a hormone.
  • the phylogenetic difference will result in a further amplification, since three to five times more of the secondary antibody will bind to chicken IgY than occurs with rabbit IgG.
  • chicken IgY is used, interference by anti- mammalian IgG antibodies can be eliminated.
  • Chicken IgY does not bind to human or bacterial Fc-receptors, such as Staphylococcal protein-A or Streptococcal protein-G .
  • Fc-receptors such as Staphylococcal protein-A or Streptococcal protein-G .
  • IgY can be used for microbiological assays without the risk of interference by Fc-receptors.
  • the present invention encompasses processes for producing desired antibodies in an avian species using polynucleotide vaccination, processes for determining the proteomics profile of a set of pre-selected DNA sequences isolated from a bio-sample and processes for identifying physiologically distinguishable markers associated with a physiologically abnormal biosample using the antibodies generated by the polynucleotide vaccination of an avian species.
  • Antibody arrays and integrated databases for identification of genes and proteins, and their uses in proteomics studies and other fields are further provided.
  • the present invention provides a process for producing antibodies to an antigen in an avian species, which comprises: 1) delivering to said avian species a DNA sequence encoding said antigen operatively linked to a promoter, said promoter being capable of directing expression of said antigen in said avian species, or a mRNA sequence encoding said antigen, in a amount sufficient to induce detectable production of said antibodies to said antigen; and 2) recovering said antibodies from said avian species.
  • the avian species to be vaccinated is selected from the group consisting of a chicken (Gallus), a quail (Coturnix), a turkey (Meleagris gallopavo), a duck, a goose and a Japanese quail (Coturnix japonica). More preferably, the avian species to be vaccinated is a chicken or a quail.
  • chicken examples include, but are not limited to, Gallus (G. domesticus), chick and hen. Such synonyms are encompassed by the present invention. For consistency, and not for limiting the scope of the presently claimed invention, only the name “chicken” is used herein.
  • keeping chickens in cages under laboratory conditions is advantageous, in that the chickens can be readily located and their health can be easily monitored.
  • the eggs can be identified unequivocally.
  • Antibodies can be produced by using chickens bred for commercial egg production as well as by using chickens which have been bred free from specific pathogens ( SPF chickens ). It is preferable to use chickens used for breeding purposes than those used for egg production, because the health status of breeding animals is often better controlled.
  • SPF chickens can be obtained from some commercial suppliers in Europe (for example, F.E. Lohma n, Cuxhaven, Germany) and in the United States (for example,
  • FCA does not influence egg production as much as the antigen itself, as has been shown, for example, for substances from Ascaris suum (.Schade, R., Burger, W., Sch ⁇ neberg, T., Schniering, A., Schwarzkopf, C, Hlinak, A. & Kobilke, H. (1994).
  • Avian egg yolk antibodies The egg laying capacity of hens following immunization with antigens of different kinds, origin, and the efficiency of egg yolk antibodies in comparison to mammalian antibodies. Alte ⁇ iativen zu Tierexperimenten 11 : 75-84 ); the results of this study indicated that the egg laying capacity is influenced primarily by events other than immunization.
  • the DNA or mRNA sequence can be delivered to the interstitial space of tissues of the animal body, including those of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
  • Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers or organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation of the lymph fluid of the lymphatic channels.
  • the DNA or mRNA sequence can be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression can be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts.
  • the DNA or mRNA sequence is delivered directly to a tissue of the avian species.
  • the DNA or mRNA sequence is delivered directly to muscle, skin or mucous membrane. Delivery to the interstitial space of muscle tissue is preferred because muscle cells are particularly competent in their ability to take up and express polynucleotides.
  • the DNA or mRNA sequence can be delivered directly to a tissue of the avian species by injection, by gene gun technology or by lipid mediated delivery technology.
  • the injection can be conducted via a needle or other injection devices.
  • the gene gun technology is disclosed in U.S. Patent No. 5,302,509 and the lipid mediated delivery technology is disclosed in U.S. Patent No. 5,703,055, the contents of which are incorporated herein by reference.
  • the DNA or mRNA sequence is delivered to a cell of the avian species and said cell containing the DNA or mRNA sequence is delivered to a suitable tissue of the avian species.
  • the DNA or mRNA sequence is delivered to a blood cell of an avian species. More preferably, the DNA or mRNA sequence is delivered to a spleen B cell of an avian species.
  • the DNA or mRNA sequence can be delivered to the cells of an avian species by a number of methods (see generally Koprowski & Weiner, DNA vaccination/ genetic vaccination, 1998. Springer-verlag Berlin Heidelberg) including Ca 3 (PO 4 ) 2 -DNA transfection (Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press, 1989), DEAE dextran-DNA transfection (Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y.
  • the gold-particle based gene gun delivery is the preferred method for delivering the DNA or mRNA sequences (U.S. Pat. No. 5,302,509).
  • Bio-Rad helios gene gun system is used in the DNA vaccination procedure. (BIO-RAD Inc. New England).
  • the helios gene gun is a convenient, hand-hold device that provides rapid and direct gene transfer in vivo.
  • the device employs an adjustable, helium pulse to sweep DNA coated gold microcarriers from the inner wall of a small plastic cartridge directly into the target cells.
  • the tubing prepstation and tubing cutter provide a simple way to prepare 50 cartridge "bullets" at a time.
  • a DNA sequence encoding the antigen operatively linked to a promoter, which is capable of directing expression of the antigen in the avian species is delivered.
  • the DNA sequence to be delivered is a plasmid.
  • the promoter to be used can be an endogenous promoter of the avian species such as chicken actin promoter.
  • the promoter can be an exogenous promoter, such as a viral promoter, which is capable of directing expression of the antigen in avian species.
  • the viral promoter is RS V LTR, MPS V LTR, SV40 IEP, CMV IEP, metallothionein promoter (U.S. Patent No. 5,703,055) or spleen necrosis virus LTR (SNV LTR) (U.S. Patent No. 5,703,055).
  • the DNA sequence used to vaccinate the avian species further comprises a sequence that directs secretion of the encoded antigen in the avian species.
  • the secretion-directing sequence is a leader sequence.
  • the leader sequence is an endogenous leader sequence of the avian species such as the leader sequence of VH1 of chicken IgY (Kabat et al., Sequences of Proteins of Immunological Interests, 1983, U.S. Department of Health and Human Services, Washington, D.C.), chicken SPARC (GenBank Accession No. L24906; Bassuk et al., Eur. J. Biochem., 1993, 2180 ⁇ :117-127), chicken serum albumin (GenBank Accession No.
  • leader sequence Although endogenous avian leader sequence is preferred, other types of leader sequences can be used in the present invention.
  • cell-membrane-directing sequence of any known membrane proteins can used. Examples of such cell-membrane-directing sequence include, but are not limited to, that of IL-1, CD4 and MHC.
  • a mRNA sequence encoding the antigen is delivered.
  • the polynucleotide material to be delivered according to the present invention can take any number of forms, and the present invention is not limited to any particular polynucleotide coding for any particular polypeptide.
  • the DNA sequence or the mRNA sequence encoding the antigen is not contained and delivered in a viral vector , such as a viral vector derived from an adenovirus.
  • substantially naked DNA sequence or mRNA sequence are used as immunogens.
  • both DNA and RNA can be synthesized directly when the nucleotide sequence is known by a combination of PCR cloning and fermentation. Moreover, when the sequence of the desired polypeptide is known, a suitable coding sequence for the polynucleotide can be inferred.
  • plasmids may advantageously comprise a promoter for a desired RNA polymerase, followed by a 5' untranslated region, a 3' untranslated region, and a template for a poly A tract. There should be a unique restriction site between these 5' and 3' regions to facilitate the insertion of any desired cDNA into the plasmid.
  • the plasmid is linearized by cutting in the polyadenylation region and is transcribed in vitro to form mRNA transcripts.
  • These transcripts are preferably provided with a 5' cap.
  • a 5' untranslated sequence such as EMC can be used which does not require a 5' cap.
  • the mRNA can be prepared in commercially-available nucleotide synthesis apparatus.
  • mRNA in circular form can be prepared.
  • Exonuclease-resistant RNAs such as circular mRNA, chemically blocked mRNA, and mRNA with a 5' cap are preferred, because of their greater half-life in vivo.
  • one preferred mRNA is a self-circularizing mRNA having the gene of interest preceded by the 5' untranslated region of polio virus (U.S. Patent No. 5,703,055). It has been demonstrated that circular mRNA has an extremely long half-life (Harland & Misher, Development, 1988, 102:837-852; Pelletier & Finberg, Nature, 1988, 334:320- 325. This material may be prepared from a DNA template that is self-splicing and generates circular "lariat" mRNAs, using the method of Been & Cech, Cell, 1986, 47:206- 216. The contents of these articles are hereby incorporated herein by reference.
  • the present invention includes the use of mRNA that is chemically blocked at . the 5' and/or 3' end to prevent access by
  • RNase This enzyme is an exonuclease and therefore does not cleave RNA in the middle of the chain.
  • Such chemical blockage can substantially lengthen the half life of the RNA in vivo.
  • Two agents which may be used to modify RNA are available from Clonetech Laboratories, Inc. Palo Alto, Calif: C2 AminoModifier (Catalog #5204-1) and Amino-7- dUTP (Catalog #K1022-1). These materials add reactive groups to the RNA. After introduction of either of these agents onto an RNA molecule of interest, an appropriate reactive substituent can be linked to the RNA according to the manufacturer's instructions. By adding a group with sufficient bulk, access to the chemically modified RNA by RNase can be prevented.
  • a chicken is vaccinated and the antibodies are recovered from egg yolk of the chicken.
  • the antibodies are purified from the egg yolk by ammonium sulfate precipitation, by polyethylene glycol 6000 precipitation or by caprylic acid precipitation.
  • immunoglobulin isolated from egg yolk of an avian species, e.g., chicken
  • IgY immunoglobulin
  • other Ig classes are present, but only in negligible amounts.
  • IgY is identical to the major Ig found in serum, but it is different from mammalian IgG (see Schade et al., ATLA, 19: 403-419) for detailed comparison between avian IgY and mammalian IgG).
  • IgG low molecular weight
  • the heavy (y) chain of IgG consists of four domains: the variable domain (VH) and three constant domains (Cyl, Cy2 and Cy3).
  • the Cyl domain is separated from Cy2 by a hinge region, which gives considerable flexibility to the Fab fragments.
  • the heavy chain of IgY (v) has a MW of 65,000 Da, does not have a hinge region, and possesses four constant domains (Cvl - Cv4) in addition to the variable domain.
  • a particularly efficient method consists of two successive precipitations in PEG, by using 3.5% PEG to remove fatty substances, and then 12% PEG to precipitate the IgY.
  • An improvement of this method incorporates an emulsification step, adding one volume of chloroform to one volume of egg yolk, rather than using the 3.5% PEG precipitation step (18, 19). It is generally assumed that about 100 mg of IgY can be recovered per egg yolk.
  • IgY purification kits available such as Promega Inc ( Madison Wisconsin USA ) EGGstract IgY purification system ( Cat #: G1531) . from PIERCE Inc. ( ROCKFORD, IL , USA ).
  • EGGCELLENT chicken IgY Purification Kit Cat #: 44918 )
  • the AmS concentration can be increased stepwise to 50% (e.g., 0 to 10%, 10 to 20%, 20 to 25%, 25 to 30%, 30 to 40%, 40 to 50%). After an incubation period at room temperature, the solution is centrifuged. The pellet is dissolved in phosphate-buffered saline (PBS) containing sodium azide (NaN 3 ) to avoid microbial growth, and the supernatant is used for the next precipitation step.
  • PBS phosphate-buffered saline
  • NaN 3 sodium azide
  • the precipitation is carried out by stirring solid polyethylene glycol 6000 (PEG) into the yolk solution.
  • PEG polyethylene glycol 6000
  • the polyethylene glycol concentration can be increased stepwise to 12% (e.g., 0 to 2%, 2 to 4%, 4 to 6%, 6 to 8%, 8 to 10%, 10 to 12%).
  • the solution is centrifuged. The pellet is dissolved in PBS containing NaN 3 . The supernatant is collected and used for the next precipitation step.
  • the yolk solution is diluted with acetate buffer.
  • Caprylic acid (CA) is stirred into the solution to a final concentration of 0.02, 0.05, 0.1, 0.5, or 1%. After an incubation period at room temperature, the solution is centrifuged. The pellet is dissolved in PBS containing NaN 3 .
  • a chicken is vaccinated and the antibodies are recovered from antibody-producing B cells of the chicken, preferably from spleen B cells.
  • the presently claimed processes can be used to generate antibodies to any protein or peptide antigens, the presently claimed processes can preferably be used to generate antibodies to secreted protein or peptide antigens.
  • the present invention provides a vector for expressing genes in avian and bacterial cells, which comprises the plasmid depicted in Figures 3A & 3C, respectively; and a vector for immortalizing chicken antibody-producing cells, which comprises the plasmid depicted in Figure 12.
  • the present invention provides a process for producing a monoclonal antibody to an antigen in a chicken, which comprises: 1) delivering to said chicken a DNA sequence encoding said antigen operatively linked to a promoter, said promoter being capable of directing expression of said antigen in said avian species, or a mRNA sequence encoding said antigen, in a amount sufficient to induce detectable production of said antibodies to said antigen; 2) removing at least a portion of antibody-producing cells from said chicken; 3) immortalizing said removed antibody-producing cells; 4) propagating said immortalized antibody-producing cells; and 5) harvesting the monoclonal antibody produced by said immortalized antibody-producing cells.
  • any antibody-producing cells can be removed in step 2) of the above process.
  • the antibody-producing cells are removed from spleen or bursa in step 2).
  • the chicken spleen B cells can be immortalized by any methods known in the art.
  • the chicken spleen B cells are immortalized by fusing with cells of a chicken B lymphoblastoid cell line.
  • Examples of chicken B lymphoblastoid cell line include, but are not limited to, HU3R27, HU3R27N and R27H4.
  • HU3R27, HU3R27N and R27H4 are disclosed in Nishinaka et al., J. Immunological Methods, 1991, 139:217-222, the content of which is incorporated herein by reference.
  • chicken B lymphoblastoid cell clones such as HU3R27, HU3R27N and R27H4, are fused with spleen cells from immunized chickens at a certain parental cell/lymphocyte ratio, e.g., 1:5 at room temperature (RT) with polyethylene glycol 6000 and poly-L-arginine in PBS.
  • the fused cells are gently washed, suspended in IMDM supplemented with FBS and plated in 96-well culture plates at the density of 3 x 10 5 spleen cells per well based on cell counts before fusion.
  • HAT medium is added to each well, and kept for 10-14 days in the same medium with repeated medium change at intervals of 2-3 days. After 10-14 days, culture supernatants from these wells are used for identification of antibody-secreting hybridomas.
  • Cloning can be performed by a soft agar culture method.
  • Growing hybridoma cells are distributed to 60 mm plates in soft agar medium containing IMDM, Noble agar (Difco), EBS and conditioned medium from a parental cell culture (Id.).
  • the soft agar plates are allowed to cool at room temperature and then incubated at about 38°C in a CO 2 incubator. Visible colonies are individually removed from the soft agar and adapted to growth in liquid medium.
  • the chicken spleen B cells are immortalized by oncogene transformation.
  • the oncogene used in transformation is mutant chicken p53 oncogene or Ras oncogene.
  • the DNA sequence or the mRNA sequence encoding the antigen is not contained and delivered in a viral vector, such as a viral vector derived from an adenovirus.
  • a viral vector such as a viral vector derived from an adenovirus.
  • substantially naked DNA sequence or mRNA sequence are used as immunogens.
  • the present invention provides a process for assessing the proteomics profile of a set of pre-selected DNA sequences isolated from a bio-sample, which process comprises: 1) cloning each of said DNA sequences into a dual-expression vector that is capable of expressing said DNA sequences in avian cells, non-avian cells or in vitro expression systems; 2) delivering said DNA sequence in said dual-expression vector formed in step 1), or mRNA or protein encoded by said DNA sequence, or a mixture thereof, to an avian species in an amount sufficient to induce detectable production of antibodies to an antigen encoded by said DNA sequence, and recovering said antibodies from said avian species; 3) delivering said DNA sequence, or mRNA encoded by said DNA sequence, or a mixture thereof, which is delivered to said avian species in step 2), to said non-avian cells, and recovering proteins or peptides encoded by said DNA sequence from said non-avian cells, or expressing and recovering proteins or peptides encoded by said DNA sequence in said in vitro
  • the set of pre-selected DNA sequences used in the process is a cDNA library.
  • the bio-sample being analyzed is of human origin.
  • the dual-expression vector is the plasmid depicted in Figure 3A or Figure 3C is used.
  • Any avian species can be used in the present processes.
  • chicken, quail, turkey, duck or goose can be used.
  • chicken or quail is used.
  • Any non-avian cells can be used for producing proteins or peptides encoded the preselected DNA sequences. Such proteins or peptides can be used as immunogens in the avian species to generate the desired antibodies and/or used in the characterization the antibodies generated from the above described processes.
  • Animal, plant, fungus and bacterium cells can be used, provided that the promoters and the non-avian cells used are compatible, i.e., the promoters can direct expression in the selected non-avian cells.
  • well-established cells, cell lines and strains are used. For example, for mammalian cells, CHO or 293 cells are preferred; for insect cells, Sf9 or High Five cells are preferred; for yeast cells, S. cerevisiae cells are preferred; and for bacterium cells, E.coli cells are preferred.
  • the DNA sequence or the mRNA encoded by the DNA sequence is used as immunogens delivered to the avian species.
  • the DNA or mRNA sequence can be delivered directly to a tissue of the avian species.
  • the DNA or mRNA sequence is delivered directly to muscle, skin or mucous membrane.
  • the DNA or mRNA sequence can be delivered by injection, by gene gun technology or by lipid mediated delivery technology.
  • the DNA or mRNA sequence can be delivered to a cell of the avian species and said cell containing the DNA or mRNA sequence is delivered to a suitable tissue of the avian species.
  • the DNA or mRNA sequence can be delivered to blood cells or spleen B cells.
  • the DNA or mRNA sequence can be delivered to avian cells via Ca 3 (PO 4 ) 2 -DNA transfection, DEAE dextran- DNA transfection, electroporation, transfection using "LIPOFECTIN”TM reagent, gene gun technology or viral gene delivery system.
  • the DNA sequence or the mRNA sequence encoding the antigen is not contained and delivered in a viral vector, such has a viral vector derived from an adenovirus.
  • substantially naked DNA or mRNA sequence can be used as immunogen.
  • the protein encoded by the DNA sequence is used an immunogen and delivered to the avian species to generate antibodies. Any known methods for generating antibodies using protein or peptide immunogens can be used.
  • fusion proteins containing the protein or peptide immunogens such as GST, His-tag, intein and CBD based fusion proteins can be used. Fusion proteins with thermally- responsive elements can also be used.
  • the antibodies generated in the avian species can be recovered by any methods known in the art.
  • the antibodies are recovered from egg yolk of the avian species such as chicken or quail.
  • the antibodies are purified from the egg yolk by ammonium sulfate precipitation, by polyethylene glycol 6000 precipitation or by caprylic acid precipitation.
  • the antibodies can also be recovered from the antibody- producing B cells of the avian species such as chicken.
  • the antibodies generated in the avian species can be characterized by the immunoreactions between the antibodies and their protein and peptide antigens.
  • immunoreactions include, but are not limited to, immunoblotting, immunoprecipitation or in situ immunostaining.
  • the immunoreactions can be conducted to determine the existence, quantity, subcellular location or tissue expression specificity of proteins or peptides encoded by the set of pre-selected DNA sequences in evaluating proteomics profile of the set of pre-selected DNA sequences in the bio-sample.
  • the present processes can be used to assess proteomics profile of any biosamples. For example, The present processes can be used to assess proteomics profile of a physiologically normal or abnormal biosample.
  • the biosamples can be derived from animals including humans, plants, fungi, bacteria and viruses.
  • the biosamples can be derived from cells when such cells are in a particular stage of a biological cycle, e.g., a particular phase of cell cycle.
  • the biosamples can be derived from particular tissues or organs, or from particular developmental stage, e.g., fetal cells.
  • the cDNA library encodes secreted proteins or peptides in the bio-sample.
  • the DNA sequence can further comprise a sequence that directs secretion of the encoded antigen in the avian species.
  • a secretion-directmg sequence is a leader sequence.
  • the leader sequence used is an endogenous leader sequence of the avian species.
  • exemplary leader sequences include the leader sequence of chicken IgY, chicken SPARC, chicken serum albumin, or chicken tissue-type plasminogen activator (tPA) and the leader sequence of IL-1, CD4 and MHC.
  • a process for identifying physiologically distinguishable markers associated with a physiologically abnormal bio-sample comprises: 1) assessing proteomics profile of said physiologically abnormal bio-sample through the above described process; 2) assessing proteomics profile of a comparable physiologically normal bio-sample through the above described process; and 3) comparing the proteomics profile obtained in step 1) with the proteomics profile obtained in step 2) to identify- physiologically distinguishable markers associated with a physiologically abnormal biosample.
  • the present invention further encompasses multi-functional gene expression vectors (pMFGEV) that are designed for rapid cloning and/or transferring target gene fragment between different vectors for functional study or can be used in different host cells such as mammalian, bacterial and insect cells.
  • pMFGEV multi-functional gene expression vectors
  • the pMFGEV vectors must contain sequences that are capable of directing gene expression in the desired avian cells and non-avian cells or in vitro expression systems.
  • the pMFGEV vector can have triple promoters which can express the same target gene by transformation or transfection into mammalian, bacterial and insect cells.
  • chicken ⁇ -actin promoter can be used for avian cell expression
  • plO promoter can be used for insect cell expression
  • T 7 promoter can be used for bacterial expression.
  • Vectors contain such triple promoters are known in the art and are commercially available, e.g., pTriEx-1 from Novagen Inc (Madison, WI, USA).
  • the pMFGEV vectors can also include sequences for facilitating site-specific homologous recombination, baculoviral recombinant element or Ad5 ITRs, and/or sequences for facilitating viral packagings, such as retroviral LTRs.
  • Such pMFGEV vectors can be used to facilitate homologous recombination among different vectors and to generate packaged viral particles, e.g., baculoviral or adenoviral particles, for high-efficiency gene transferring.
  • Figure 16 illustrates an exemplary pMFGEV vector that can be used in the processes or methods described herein.
  • the exemplary pMFGEV vector carries its own
  • ColE ori fragment for replication in bacterial cells and EBV OirP DNA fragment for maintaining episomal DNA replication after the vector is transfected into mammalian cells.
  • This vector carries the Site-Specific Recombinant Element
  • SSRE just downstream of promoter region.
  • the inclusion of SSRE can dramatically simplify gene cloning procedure for inserting a target gene into this vector.
  • SSRE element can be used including the SSRE fragment derived from well characterized systems such as Int or FLP site-specific recombination pathways (See Landy, Current
  • WO 99/21977 discloses vectors containing SSRE element, which can transfer a target gene in the defined orientation using modified lambda Int site-specific DNA recombinant mechanism.
  • cDNA library is constructed by flanking the modified lambda Int site-specific fragment and DNA sequenced
  • selected target DNA fragment can be transferred directly from cDNA library vector into pMFGEV vector by mixing the two vectors with suitable reconmbinases such as Gateway BP clonase Mix or Gateway LR clonase mix Cat # 11789-013 or 11791-019 and the enzymes made by Life Technologies Inc. MD. USA.
  • a vector for immortalizing chicken antibody-producing cells is further provided, which vector comprises the plasmid depicted in Figure 12.
  • the present invention provides a method for selecting and constructing a set of DNA sequences to generate antibodies against proteins or peptides encoded by such DNA sequences, which method comprises: (1) selecting specific tissue sample of interest; (2) extracting mRNA from the selected sample; (3) performing cDNA synthesis, preferably using modified RNA normalization procedure; (4) fractionating the synthesized cDNA, preferably by gel electrophoresis; (5) constructing a cDNA library in the vector that can express the cDNA in both avian cells and bacterial cells, such as the pS&DV depicted in Figure 3A and the pS&DV-S depicted in Figure 3C; (6) establishing the fractionated master cDNA library; (7) conducting quality assurance analysis of the master cDNA library; (8) purifying the cloned cDNA; (9) determining the sequence of the cloned cDNA; and (10) conducting bioimformatic analysis of the DNA sequence data to select the set of the DNA sequences for further DNA vaccination.
  • the tissue sample can be obtained from either fresh or frozen sources.
  • the tissue sample can be obtained from human, animal, plant or microbe. Selection of specific tissue will be determined in each specific study. For example, if one desires to obtain human liver specific gene, one could use human adult liver or fetal liver tissue as the resource for mRNA extraction. In a preferred embodiment, the tissue is used as fresh as possible.
  • mRNA extraction kits such as the kit from Life Science BRL (Gaithersburg, MD) and Promega Inc. (Madison, WI), can be used.
  • the tissue sample can be rapidly frozen in liquid nitrogen, grounded and resuspended in RNA extraction buffers such as 4M guanidine solution.
  • RNA extraction buffers such as 4M guanidine solution.
  • mRNA can be extracted directly from the tissue using lysis buffer, and the extracted mRNA can be further purified on an ion exchange column, such a the column from Qiagen (Chatsworth, CA).
  • junky clones are (a) clones that consist exclusively of poly(A) tails; (b) clones that contain very short cDNA inserts; (c) clones that contain nothing but the 3' half of the NotI-oligo(dT)18 primer used for synthesis of first strand cDNA ligated to the adopter; and (d) chimerical clones.
  • the classical procedure for normalization and subtraction of RNA for cDNA synthesis can be used (Bonaldo et al., Genome Research, 1996, 6:791-806; Neto et al., Gene, 1997, 186:135-142).
  • the burden of large-scale DNA sequencing is the repeated sequencing of the same clones multiple times.
  • Using non-amplified cDNA library can help to improve the DNA sequence and checking first-strand cDNA synthesis efficiency, which can also be the index for determining the quality of reverse-transcription reaction (Bodescot & Brison, BioTechniques, 1997, 22(6): 1119-1125).
  • the total cDNA products can be fractionated by gel electrophoresis, such as 0.8% to 1% agarose gel. Then the desired size of cDNAs can be pooled and extracted before ligating into the cloning vectors, such as the vectors from Qiagen (Chatsworth, CA).
  • the collected length of cDNAs can vary such as every 0.5 kb as the region for the one pooled sample. Those pooled cDNAs can then be inserted into vector in the different ligation tubes designated for different transformation experiments. In the mixed cDNA library, the full-length cDNA often resides in a complex background of small cDNA mixture. This fractionation procedure for cDNA preparation can generate a sub-population of cDNA library.
  • the designated length of cDNA clones will also help to identify the full-length clone from the subpopulation of cDNA once the 3'EST sequence are known and the size of gene transcript is obtained from the standard RNA Northern Blot experiments (Chenchik et al., Bio/Techniques, 1996, 21 :526-534). This procedure is applicable to the construction a variety of cDNA libraries from different tissue samples.
  • the following illustrates procedures for constructing cDNA library using a dual- expression vector, such as the pS&DV and pS&DV-S depicted in Figure 3A and 3C, respectively.
  • the purpose for directly cloning cDNA insert into a dual expression vector is to enable conducting DNA vaccination in an avian species and to expressing encoded protein or peptide antigen in bacteria with the same DNA.
  • This dual functional vector carries those fragments for avian cell gene expression such as endogenous avian promoters, or viral promoters such CMV promoter, SV40 intron and SV40 polyadenylation site.
  • the vector also carries T7 RNA polymerase promoter expression system in which the cloned gene will express in E.
  • coli strain that carries T7 RNA polymerase such as BL21 (DE3) (Studier et al., Methods in Enzymol, 1990, 185:60-89). Due to the codon preference between human and E.coli, some human cDNAs or genes may be expressed at low level in commonly used E.coli host cells. By adding three new modified human gene codons commonly used in tRNA gene into E.coli strain, this newly developed E.coli strain (BL21 CodonPlus-RIL), which is suitable for the human cDNA or gene expression, is commercially available, e.g., from Strategene Inc ( Cat #: 230245 ).
  • Strategene Inc Cat #: 230245
  • the cDNA fragments can be directionally inserted into the vector by digesting the cDNA at both ends with different restriction enzymes, such as Pad for 5' and Notl for 3' end. Cloning the cDNA with correct orientation will ensure the expression of the gene. Considering that most of cDNA fragments may lack 5' region or translation initiation code (ATG) in its fragment, the artificial ATG has been created in the vector to enhance the protein expression level in the bacterial by adjusting the distance of Shine-Dalgarno/Kozak consensus sequence between the ATG. There are multiple cloning sites located just downstream of ATG. And three sets of different open read frames (ORF) have been constructed in the vector.
  • ATG translation initiation code
  • pS&DV-S contains a chicken IgY leader sequence, which can direct secretion of the proteins or peptides encoded by the cDNA inserts in chicken cells.
  • the following illustrates procedures for constructing a master cDNA library consisting of subpopulation of fractionated cDNA clones.
  • the vector and cDNA ligation mixture can be efficiently transformed into bacterial cells such as HB101 cells using standard procedure, preferably by electroporation, which are available from different commercial vendors, such as Life Science BRL (Gaithersburg, MD).
  • the clones can be picked up and then transferred into 320 well plate which contains frozen reserve solution such as 15% glycerol (Ausubel et al, Current Protocols In Molecular Biology, New York, John Wiley and Sons, 1995; and Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press).
  • frozen reserve solution such as 15% glycerol (Ausubel et al, Current Protocols In Molecular Biology, New York, John Wiley and Sons, 1995; and Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press).
  • the separated clones for total library should preferably be at least 10 times more than potential human gene numbers. Preliminary data from published data base indicate that there may be approximately 100,000 functional genes in human genome. So for each tissue-specific cDNA library, about 1 million clones have to be picked up for further DNA sequencing analysis.
  • the automatic clone pick-up systems are available from different commercial
  • the following illustrates procedures for conducting quality assurance analysis of the master cDNA library.
  • quality assurance analysis before large-scale DNA sequencing will ensure the desired outcome in a cost efficient fashion.
  • a portion of cDNA library can be analyzed and then the data be used for determining the quality of the total cDNA library.
  • about 1,000 clones can be randomly picked up from the library, DNA sequencing can be performed using a specific primer for cDNA 5' sequence. Analyses of such limited sequencing data will give useful information such as the gene distribution pattern, the length of inserted gene and percentage of vector self-ligation.
  • clones can be cultured on a plate(s) and be replicaed to nitrocellulose membrane and be screened by DNA hybridization using housekeeping gene sequence as the probe.
  • the probe For example, if the cDNA library is derived from liver tissue, /3-actin and/or albumin nucleotide sequences can be used as the probe.
  • the probes can be labeled by any techniques known in the art. In a preferred procedure, the probe is labeled using the random primer method (Feinberg & Vogelstein, Analyt.
  • 32 P-dNTP is incorporated into a random primer labeling reaction using a kit such as the DECprime II DNA labeling kit (Ambion,
  • nonisotopic labeling agents can be used (Kricka, ed., Nonisotopic Probing,
  • the hybridization results can be used to review the background contamination of housekeeping genes in the normalized cDNA library. Since most of the housekeeping genes occur at about the same rate in cDNA library generally, the hybridization rate for housekeeping genes can be used to determine the quality of the cDNA library. The quality of the cDNA library can also be determined by PCR-based procedures. (Pacchioni et al., BioTechniques,
  • PCR amplifications are carried using the housekeeping genes, such as /3-actin gene, as the 3' DNA specific oligo and the T7 promoter oligo as the 5' primer in the randomly picked cDNA library clones. Subsequent detection for presence or absence of PCR products (+/- scores) is carried out either by gel electrophoresis or by internal oligonucleotide hybridization. The PCR amplification results will not only reveal the percentage of the housekeeping gene's presence in the cDNA library, but can also be used to determine the average length of the cDNA insert.
  • the PCR amplification reaction of the random clones of the cDNA library can be conducted using commercially available reagents or kits, such as the ones produced by Origene
  • the cloned plasmid DNA can be purified by any methods known in the art.
  • the automatic plasmid purification equipment such as the Quiagen Inc automation system 9600 (Valencia, CA) can be used to provide highly purified DNA template for subsequent DNA sequencing analysis.
  • cDNA Clones can be used for PCR amplification and nest-PCR again to provide DNA sequencing template. Since the sequence is known, the two pare of primers for PCR can be easily standardized for all of the clones in the library.
  • DNA sequences can be determined by any methods known in the art.
  • each randomly selected clone is purified from a cDNA library, a DNA sequencing template is prepared.
  • This template is sequenced by the dideoxy method, preferably using an automated DNA sequencer, such as an A. L. F. (Pharmacia Biotech, Piscataway, N. J) or an ABI/373 or ABI/377 (Applied Biosystems, Foster City, Calif).
  • an automated DNA sequencer such as an A. L. F. (Pharmacia Biotech, Piscataway, N. J) or an ABI/373 or ABI/377 (Applied Biosystems, Foster City, Calif).
  • a "walking" phase takes additional reading from selected clones by use of custom primers.
  • oligonucleotide sequences are generally designed to preferentially detect sequences that are related to the ends of genes in the previous DNA sequence database. This selective bias can be achieved either by manually reading of sequence or by examination of the sequences to be compared.
  • these oligonucleotides can be ordered from a DNA synthesis service such as the Research Genetics, (Huntsville, AL). Alternatively, the oligonucleotides can be synthesized on a DNA synthesizer, e.g., on the Applied Biosystems (Foster City, CA).
  • the DNA sequencing reaction products can be separated by electrophoresis, preferably on polyacrylamide gels using fluorescence detection.
  • Other DNA size separation technologies such as ultrathin gel slabs (Kostichka et al., Bio/Technology, 1992, 10:78-81), capillary arrays (Mathies & Huang, Nature, 1992, 359:167-169), and mass spectrometry (Wu et al., Rapid Commun. Mass Spectrom., 1993, 7:142-146), can also be used.
  • DNA sequencing analysis without using gel electrophoresis has also been done by hybridization methodologies (Drmanac et al., Science, 1993, 260:1649-1652; Southern et al., Genomics, 1991, 13:1008-10017).
  • Another approach is the base addition sequencing strategy (BASS), which uses synchronized DNA polymer construction to determine the sequence of unknown DNA templates (U.S. Patent No. 5,302,509; WO 93/21340; and WO 91/06678).
  • sequences of the selected clones by "walking" procedure can be assembled into the complete cDNA sequence of the inserted DNA by matching overlaps.
  • Computer programs are available for these tasks (e.g., Rodger Staden programs, Cambridge, UK; DNAStar, Madison, Wis.).
  • similarity and homology searches can be conducted in relevant sequence databases (e.g., GenBank, Bethesda, Md.; EMBL, Cambridge, UK; Phil Green's GENEFINDER, Seattle, Wash) to identify genes and repetitive elements, to infer function, and to determine the sequence's relation to other parts of the genome and cell (Gonzalez & Sylvester, Genome Research, 1997, 7:65-70).
  • sequences of the selected clones by universal primer from 5' of mserted DNA can be firstly analyzed using specific computer program. For example, similarity and homology searches can be conducted (GenBank,
  • the full-length cDNA coded putative protein can be further analyzed such as for functional domain searching.
  • the analysis data can be categorized into computer database.
  • Other experiments, such as looking for the DNA transcription control elements after function of the cDNA is mapped, can also be conducted (Fickett & Hatzigeorgious, Genome Research, 1997, 7:861-878).
  • the processes described in ⁇ A and B. can be used to generate desired antibodies, whether polyclonal or monoclonal ones, against the proteins or peptides encoded by such selected DNA sequences.
  • the DNA sequences used in the DNA vaccination are also delivered into competent bacteria cells to produce the encoded proteins or peptides, which can be used in characterizing the antibodies generated by the DNA vaccination.
  • the bacteria cells are competent E. coli. cells.
  • the dual-expression vector depicted in Figure 3A or 3C is used in delivering the DNA sequence into bacteria cells.
  • the proteins encoded by the delivered DNA sequences can be expressed at high level in the presence of an inducer, e.g., IPTG.
  • DNA sequences can be delivered into bacterial cells by any methods known in the art (e.g., Ausubel et al., Current Protocols In Molecular Biology, New York, John Wiley and Sons, 1995; and in Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press).
  • methods known in the art e.g., Ausubel et al., Current Protocols In Molecular Biology, New York, John Wiley and Sons, 1995; and in Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press.
  • commercially available systems for DNA transformation such as the one from Life Science BRL (Geitesburg. MD), can be used.
  • Bacterially expressed proteins or peptides can be recovered by any methods known in the art (e.g. , Ausubel et al., Current Protocols In Molecular Biology, New York, John Wiley and Sons, 1995; and in Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press).
  • transformed bacterial clones can be picked up and grown in LB culture medium. Before harvesting the bacterial cells, an inducer such as IPTG can be added to induce the protein expression.
  • Bacterial cells can be harvested by centrifugation, resuspended directly in SDS-PAGE lysis buffer and analyzed by SDS- PAGE using commercially available system, such as the one from Bio-RAD Inc. (Hercules, CA).
  • the immunoreactions between the antibodies generated by the DNA vaccination and the bacterially expressed proteins or peptides can be analyzed by any methods known in the art (e.g., Ausubel et al., Current Protocols In Molecular Biology, New York, John Wiley and Sons, 1995; and in Sambrook et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press). Preferably, such immunoreactions are analyzed by immunoblotting.
  • the proteins and peptides to be analyzed can be transferred onto a suitable membrane, e.g., PVDF membrane, according to the procedures described in Schielen et al., Journal of Immunological Methods, 1995, 188:33-41.
  • the immunoblotting reaction can be analyzed by any methods known in the art.
  • the immunoblotting reactions are detected by commercially available system, such as the Chemiluminescence detecting system from BIO-RAD (Hercule, CA).
  • the positive results generated from immunoreaction between the antibodies and the bacterially expressed proteins or peptides only confirm that proteins or peptides are encoded by the DNA sequences isolated from bio-samples. After the antibodies are characterized by the immunoreactions between and the bacterially expressed proteins or peptides as described above, further immunoreactions between the antibodies and the biosample, from which the DNA sequences are isolated, can be conducted to determine the proteomics profile of the selected DNA sequences.
  • the DNA fragment can be inserted into three ORFs and Western Blot assay using three different antibodies can be performed.
  • any known methods can be used to analyze the immunoreactions between the antibodies and the bio-sample.
  • immunoblotting, immunoprecipitation and in situ immunostaining are used.
  • the antibody-based methods can be used in conjunction with other techniques, such as two-dimensional electrophoresis (2-DE), ultrasensitive mass spectrometry (MS), and other high-throughout functional screening assays (Persidis, Nature Biotechnology, 1998, 16:393-394), in the proteomics studies.
  • 2-DE and MS analyses include, but are not limited to, isoelectric focusing followed by mass-based separation (ISO-DALT), non-equilibrium based electrophoresis (NEPHGE), and immobilized first-dimension pH gradients (IPG-DALT) (Humphery-Smith . et al. Electrophoresis, 1997 18:12171242).
  • ISO-DALT isoelectric focusing followed by mass-based separation
  • NEPHGE non-equilibrium based electrophoresis
  • IPG-DALT immobilized first-dimension pH gradients
  • tissue immunostaining which technology is well known in the art (Feitelson & Zern, Clinics In Laboratory Medicine, W.B.Saunders Com., 1996).
  • cryosected tissue samples are used to perform the immunostaining assay because the tissue sample fixed with this method can preserve the cellular antigen structure.
  • the data from this assay may well represent the cellular protein expression pattern in the tested tissue.
  • paraffin fixed tissue sample can be used for antibody immunostaining because this type of tissue fixation preserves the tissue for long time and also can be easily collected from different medical research resources.
  • There are several techniques which can be used to improve the immunostaining sensitivity when using paraffin fixed tissue samples Lithys et al., Surgical Endoscopy., 1998, 12(2):170-176).
  • the antibodies generated by the present invention and the information obtained from analyzing the immunoreactions between such antibodies and the bio-sample can used in number of ways.
  • One such use is the generation of an antibody index and the incorporation of such antibody index into the known nucleotide sequence databases.
  • the antibody index generated by the present invention can be automatically linked to each of corresponding cDNA sequence, the Western blot data and tissue immunostaining data can be cross-referenced in the database.
  • the subcellular image generated by the immunostaining with the cDNA derived antibody can be stored, and western blot analysis data can be traced in the database for estimating the size of specific cDNA encoded- protein.
  • the antibodies generated by the present invention can also be used in the functional analysis of the proteins or peptides encoded by new cDNA sequences.
  • the information generated from cDNA derived antibodies can be categorized into group of functional index (Poustka et al., Cold Spring Harbor Symp. Quant. Biol, 1986, 51:131-139).
  • group of functional index Paneka et al., Cold Spring Harbor Symp. Quant. Biol, 1986, 51:131-139.
  • the expression level of a specific gene can be determined using very- well documented protein such GAPDH or /3-actin as internal control. Based on those leading information for a specific gene, one can design multiple- gene functional assays to further elucidate the cellular function of the gene and understand the relationship of the gene with a specific disease, if the gene is linked to a disease or a disorder.
  • the DNA sequence provides information about the long-term inherited DNA stored in the nucleus and about the physical linkage of the genes in a genomic context. However, it is also useful to know how these genes are expressed and their cellular localization.
  • cDNA libraries have been constructed to assess gene expression in particular tissues, and methods such as direct selection have been developed to map these cDNAs relative to a genome (Lovett et al., Proc. Natl. Acad. Sci., 1991, 88:9628-9632). Other methods such as exon trapping are similarly used to measure gene expression and map exons (Buckler et al, Proc. Natl. Acad. Sci., 1991, 88:4005-4009).
  • Mutagenesis is a powerful tool to study a gene's function.
  • the selected gene can be mutated and cloned into the specific vector for generating transgenic animal, such mice, and the phenotype of the transgenic animal can be used in elucidating the target gene's function in vivo (Stewart, Molecular Medicine Today, 1997, 3(3 :93; Hickset al., Nature Genetics, 1997, 16(4 :338-344).
  • the activity of the specific cDNA encoded protein can be inhibited by a variety of technologies, such as modified oligo antisense inhibition (Milner & Southen, Nature Biotechnology, 1997, 15:537-541), target sequence- specific ribozyme inhibition (Duan et al., Gene Therapy, 1997, 4:533-543) or single chain antibody (sFv) based intracellular immunization approach (Duan et al., Proc. Natl. Acad. Scz ' ., 1994, 91 :5075-5079).
  • the present invention can be used to study the proteomics of the selected cDNA sequences of such knock-out organisms.
  • the present invention provides a process for determining the proteomics profile of a set of pre-selected DNA sequences isolated from a physiologically normal bio-sample. In another specific embodiment, the present invention provides a process for determining the proteomics profile of a set of pre-selected DNA sequences isolated from a physiologically abnormal bio-sample.
  • the abnormality of such bio-sample can be permanent or temporary, and can be caused by genetic changes or otherwise.
  • the physiologically abnormal bio-sample is obtained from a subject who/that has or is known in the high risk of having any diseases or disorders.
  • nucleic acid immunogen Delivery of nucleic acid immunogen by viral gene delivery systems
  • the DNA sequence or the mRNA encoded by the DNA sequence can be used as immunogens and delivered to the avian species. Any known methods for generating antibodies using nucleic acid immunogens can be used. For example, the processes described in the above Sections A and B can be used. Alternatively, the nucleic acid immunogen can be delivered to avian species by recombinant viral gene delivery systems.
  • Retroviruses owe to their high infectivity, special structure and the capacity to be integrated readily in the form of a provirus in the genome of the host cells, have been widely used for gene delivery. Since cDNA fragment or target gene can be easily constructed into retroviral based vector for gene delivery, the avian cell infectable refroviral systems such as avian erythroblastosis retrovirus (AEV) and Spleen Necrosis Virus (SNV) can be used in the present processes for to deliver nucleic acid immunogens to the avian species.
  • AEV avian erythroblastosis retrovirus
  • SNV Spleen Necrosis Virus
  • the genome of a retrovirus in its replication-defective form is composed of an RNA molecule possessing, in the direction of the transcription (5' to 3') an identical short sequence at each end, known as R. This is followed, in order, by a single sequence known as U5, a tRNA binding site (TBS), and a non-coding sequence ("leader" sequence).
  • RNA molecule continues with a region coding for three genes, the translation products of which are essential for the replication of the virus, and which are gag (virion structural proteins), pol (reverse polymerase) and env (envelope).
  • the genome terminates, in order, with a non-coding sequence, a purine-rich sequence (PU), a single sequence known as U3, and finally the R sequence.
  • PU purine-rich sequence
  • U3 purine-rich sequence
  • R sequence The repeat end sequences (R) or single sequences (U5 and U3) peculiar to retroviruses appear to be rather well conserved in this group of viruses and contain the signals involved in the control of the expression of the viral genome.
  • helper viruses are capable of helping replication of the replication-defective viruses with foreign nucleic acid fragment, but cannot self-replicate because the helper viruses contain only the functional gag, pol and env genes without necessary cis-acting signals for its own replication.
  • the cycle of infection by a retrovirus begins with the adsorption of the virions on the surface of the host cells, followed by penetration into the cytoplasm.
  • the single-stranded viral RNA (a) is transcribed by the reverse polymerase present in the virion, to a linear copy of double-stranded DNA (b).
  • the DNA copy resulting from this reverse transcription is slightly longer than the viral RNA molecule which acts as a template for it. This difference is the result of the addition of an U3 sequence at the 5' end and an U5 sequence at the 3' end.
  • the combination, in order, of the U3-R-U5 sequences constitutes a repeat sequence at both ends of the DNA molecule, known as LTR (Long Terminal Repeat).
  • LTR Long Terminal Repeat
  • the copies of viral DNA containing one or two LTR sequences are conveyed to the nucleus where they are converted to molecules of circular shape. Some circular molecules only retain a single LTR. These molecules are then integrated in the host cell genome.
  • the viral DNA is integrated in the host cellular DNA in such a manner that it is enclosed by an LTR at each end, and then bears the name of proviral DNA or provirus.
  • the provirus acts as template for the transcription of viral RNA molecules.
  • the transcription is initiated at the R sequence of the left LTR and stops beyond the polyadenylation signal carried by the U3 or R sequence of the right LTR.
  • the RNA molecules obtained after transcription of the provirus are a reflection of the mRNA of the eucaryotic cells, "capped” by a terminal 7mG residue at the 5' end and provided with a polyadenylated sequence at their terminal 3' end. Delivery via AEV vector
  • AEV retrovirus as a vector for delivery of a foreign gene
  • vector AEV can use different forms as a result of wide selectable vector available now.
  • the description below refers more especially to AEV virus for its gene delivery into avian cell such as chicken , but the method generally relates to other retroviruses such as SNV which will described in the following section.
  • the vector AEV is employed under conditions which enable its replication and the formation of virions to take place, that is to say with a helper virus, the infection of cell culture in vitro or directly viral particle injection in vivo may be carried out with considerable efficiency, taking into account the multiplication of the infectious virions.
  • the following description illustrates insertion of a human gene or cDNA fragment into the AEV vector.
  • restriction enzymes such as EcoRI and Notl
  • specifically selected gene or random selected human cDNA fragments can be inserted into an AEV vector such as pAEV2LTRdelta (U.S. Patent No. 4,957,865).
  • All the recombinant plasmids can be characterized by restriction mapping or directly DNA sequencing.
  • the recombinant plasmid pAEV2LTRdelta can be transfected in the presence of DNA of the helper virus (pRAV2) into secondary cultures of chick embryo fibroblasts. Cells are maintained in liquid medium and subcultured at regular intervals.
  • pRAV2 helper virus
  • the culture medium is composed of DMEM (Gibco) supplemented with 10% of foetal calf serum, 2 mM glutamine, 2.2 mg/ml of sodium bicarbonate, 100 ⁇ g/ml of streptomycin and 100 iu/ml of penicillin.
  • the stocks of recombinant virus can be titrated for their drug selection according to the technique of counting of colonies. For this purpose, 1 ml of diluted viral suspension is inoculated on cultures of fresh fibroblasts. The cultures are then selected with drug such as G418. The titer of the virus is given by the number of colonies of transformation per dish multiplied by the dilution of the suspension of inoculated virus. The titer is expressed in FFU (focus forming unit) per ml. By centrifugation of viral stock, the virus can be pelleted and re-suspended in serum free cell culture medium. This viral re-suspension preparation (1 ml) can be directly used for the animal injection in the range of 10 ⁇ / ml.
  • Spleen Necrosis Virus can also be designed for the purpose of avian cell gene transduction to immunize the chicken.
  • SNV spleen necrosis virus
  • the SNV based vector for the gene delivery application is also summarized by Dounburg Ralph in the WO 00/09730 and Pathak and Vinay K in U.S. Patent No. 5,714,353.
  • SNV based vector is packaged in SNV permissive D17 dog cells, the recombinant SNV viral particle, which carries a human gene, can be used for the transduction of avian cell efficiently.
  • SNV -refroviral shuttle vectors designated DHH-N-2neo and JJ-A2neo, were deposited the American Type Culture Collection (ATCC), 13201 Parklawn Drive, Rockville, Md., 20852, and has been accorded ATCC Accession Number 97861 and 75780, respectively.
  • Adenovirus is a large and diverse family of viruses. Adenoviruses have been isolated from many living species, including man and other mammals, as well as a variety of birds, particularly chicken. One group of virus, Aviadenoviradae, which is defined by its avian host range, can infect avian cell efficiently.
  • Adenovirus vectors are capable of high level expression of carried exogenous proteins, because transcription from the major late promoter of adenovirus is very efficient and high level translation is accompanied by host protein synthesis shut-off in the late stage of viral infection, thus facilitating protein isolation.
  • human adenoviruses can replicate efficiently to very high titers (10 9 -10 10 pfu/ml) in human cells, as well as in other mammalian cells; and adenoviruses can produce their late proteins at a level that reaches 30 to 40% of total cellular proteins.
  • Adenovirus vectors can also be propagated in suspension cultures thereby demonstrating a clear potential for large-scale production.
  • Patent No. 5,518,913 includes an expression cassette comprising sequentially a transcription promoter, a high efficiency leader, at least one splicing signal, an enhancer-like sequence, a cloning site and a plurality of polyadenylation sites. According to U.S. Patent No. 5,518,913, recombinant protein production, in cells infected with the recombinant adenovirus, can approach levels as high as 15-20% of total cellular proteins and can be used as animal immunization antigen.
  • the avian based packaging cells can be generated using avian cells which are transfected with FAV CF20 viral El a gene and can be manipulated in the same way as 293 A (adherent) cells, which are derived from human kidney fibroblast transformed with Ad5 DNA and express the El A and EIB proteins constitutively, are manipulated.
  • the avian cells can be obtained from the ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories), supplemented with 10% fetal bovine serum (FBS), glutamine and antibiotics.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • FAV CF20 can be used as the parent virus in all the viral constructs, and recombinant Ad are propagated by infecting monolayer Ad packaging cells.
  • Avian help virus can be created the same way as AdRed-1, a helper-free Ad recombinant expressing the HSV-2 ribonucleotide large subunit Rl, is created (Huang et al., 1988, Virology 163:462-470).
  • Large-scale production of Ad stocks is done by infecting exponentially growing packaging cells (0.5X10 6 cells/mL) at a MOI of 10-50 PFU/cell and harvesting the infected cells at 72 hours post-infection.
  • the cell pellet is then resuspended in fresh Joklik's modified medium at a cell density of 1X10 8 cells/ml and virions are released by three to six cycles of freezing and thawing.
  • Adenovirus titers are determined by plaque assays on packaging cells. Cell counting is performed using a hemacytometer, and viability is determined by trypan blue dye exclusion.
  • All recombinant DNA molecules can be constructed by standard cloning and site- directed mutagenesis procedures and propagated in competent E.coli cells, e.g., DH5 cells.
  • the transfer vector based on FAV CF20 can be derived from FAV Ml 1 (WO 94/24268) by sequentially subcloning human cDNA fragments into the vector with compatible Notl ends.
  • the coding region of the desired human cDNA gene can be first cloned in transfer vectors at the unique Notl cloning site, downstream of the strong
  • the resulting recombinant plasmid is then rescued into the genome of the adenovirus vector by in vivo homologous recombination between overlapping sequences on the linearized plasmid and the large right-end fragment of the FAV CF20 genome, upon cotransfection of avian packaging cells.
  • This cell line constitutively expresses the avian Ad El gene product, which is essential for the helper-free propagation of FAV CF20 derived recombinants. Digestion of the help viral DNA with unique single cut prior to transfection allows for obtention of recombinant adenovirus at a frequency of 5- 20%.
  • the virions are purified through 2 consecutive CsCl gradients.
  • a step gradient is performed by pouring 8 ml of CsCl 1.4 (53 gr+87 ml of 10 mM Tris pH 7.9) into SW 27 cellulose nitrate tubes, and then very gently on top pour 56 ml of CsCl 1.2 (26.8 gr+92 ml of 10 mM Tris pH 7.9).
  • the aqueous phase containing the virions is then loaded on top of the discontinuous gradient (up to 22 ml/tube).
  • the tubes are then centrifuged at 23K for 90 minutes at 0°C.
  • the virus band is then collected by side puncture of the tubes.
  • the band is diluted 1/2 in 50 mM Tris pH 7.5, 1 mM EDTA.
  • a continuous gradient is then performed by using a gradient maker, to pour a continuous CsCl gradient in SW27 cellulose nitrate tubes using 12 ml of CsCl 1.4 and 14 ml of CsCl 1.2.
  • the diluted virus suspension (8-10 ml) is then loaded very slowly on top of the gradient.
  • the tubes are then centrifuged at 23K for 16-20 hours at 0°C.
  • the virus band is then collected by side puncture, and dialyzed against 100 volumes of 10 mM Tris pH 7.9, 1 mM EDTA (3 changes), and finally against 100 mM Tris pH 8.5 1 mM EDTA.
  • human cDNA derived protein can be done by infecting suspension cultures with the appropriate recombinant viruses.
  • Ad infections are performed by mixing exponentially growing avian cells (0.5. XI 0 6 cells/mL) with a viral inoculum corresponding to a MOI of 25-50 PFU/cell and harvesting the infected cells usually at 48 hours post-infection. Twenty four hours post-infection, the medium is replaced. Again, the infected cells are harvested 48 hours post-infection, washed twice with ice-cold PBS, pelleted, resuspended in ice-cold buffer A (50 mM Hepes pH 7.6, 2 mM DTT) and frozen at -80°C.
  • the time course of recombinant protein production by avian AD vector infected cells can be analyzed by preparing cell extracts from infected cells at various time points post-infection and subjecting them to SDS-PAGE.
  • Cell extracts are prepared from infected cells at various time post-infection either directly on petri dishes or from aliquots of suspension cultures containing between 1 and 2X10 6 cells/ml. Briefly, cells are washed twice with PBS, lyzed in 100 ml of extraction buffer (80 mM tris pH 6.8, 2% SDS, 10% glycerol), and frozen at -20°C.
  • samples are thawed, passed several times through a syringe needle or sonicated to shear the DNA, and boiled 5 minutes in standard sample buffer.
  • Protein concentration is determined in the cell extract using a calorimetric assay with BSA as standard.
  • SDS-PAGE and Western blotting can be performed using procedures known in the art. Quantification of recombinant protein in cell extracts is done by densitometry scanning of Coomassie blue-stained gels or immunoblots with immunized chicken IgY antibody.
  • proteins or peptides encoded by the DNA sequence can be used as immunogens and delivered to the avian species. Any known methods for generating antibodies using protein or peptide immunogens can be used.
  • the proteins or peptides can be chemically synthesized according to the DNA sequence encoding them, can be produced recombinantly, or can be produced by a combination of chemical synthesis and recombinant production.
  • the protein or peptide immunogens are produced recombinantly.
  • a gene or DNA fragment can be cloned into bacterial expression vectors such as PBR322, pUCl 8, etc.
  • the recombinant proteins can be expressed in bacterial strain such as HB101, DH5 ⁇
  • the recombinant proteins can be purified by conventional protein or peptide purification methods such as HPLC, HPLC, SDS-PAGE coupled with protein elusion from gel slice. The purified protein can then be used in immunizing the avian species.
  • fusion proteins containing the protein or peptide immunogens such as GST, His-tag, intein and CBD based fusion proteins can be used. Fusion proteins with thermally-responsive elements can also be used.
  • a target gene or cDNA fragment can be generated by the polymerase chain from specifically constructed cDNA library and cloned into the
  • GST-fusion expression vector such as pGEX-2T (Amersham-Phamacia Biotec, Uppsala,
  • This plasmid contains a thrombin cleavage recognition sequence (Leu-Val-Pro-Axg-Gly-Ser (Inserted gene) between a sequence encoding a glutathione-S-transferase tag and the cloning site into which the cDNA sequence is inserted.
  • the resulting plasmid, encoding the target cDNA as a fusion protein with a GST tag, can be transformed into bacterial cells after DNA sequence is confirmed.
  • the recombinant GST expression plasmid can be transformed into commonly used bacterial strains such as HB101 or BL21 ( DE3). Transformed cells are grown on LB agar plates overnight with ampicillin selection, e.g., ampicillin present in the plates at 100 ug /ml.
  • All growth can be carried out at 37°C.
  • a single colony from a plate of transformed E.coli is used to inoculate 2 ml of LB or Typ medium (16g bactotrytone, 16g bacto yeast extract, 5g sodium chloride, 2.5 g potassium dihydrogen phosphate per liter) with ampicillin present at 100 ug / ml which is established for 3 hours with aeration.
  • the colony can be inoculated into 250 ml of LB medium made with 10 g bacto-tryptone, 5 g yeast extract and 5g sodium chloride and supplemented with ampicillin at 100 ug/ml and left to stand overnight. The next day, 25 ml aliquots of this culture is inoculated into 1 liter flask.
  • the 1 liter culture is frown with aeration to mid-log-phase growth (an optical density (Abs600) of 0.6-0.8 AU), where upon expression from the plasmid is induced with 0.4 mM isopropyl-b-D thioglactopyranoside (IPTG).
  • IPTG isopropyl-b-D thioglactopyranoside
  • the cell pellet either immediately or after storage at -80°C is re-suspended in 100 ml cold phosphate buffer saline (PBS).
  • PBS cold phosphate buffer saline
  • the bacteria can be lysed either by sonication or chemical lysis buffer such as PIERCE Inc B-PER lysis buffer. After sonication, 5 ml of 20% Triton X 100 ( Sigma Inc) is added to give a final concentration of 1% and leave this mixing at 4°C for 30 minutes, preferably with gentle agitation. The entire preparation is centrifuged at 12,000 X g for 30 minutes at 4°C. Supernatant is retained and pellet is discarded.
  • Recombinant GST-fusion protein purification Fusion protein can be purified using commercially available kit such as PIERCE Inc GST Orientation Kit (Cat # 78201), or can be prepared using the following procedure:
  • Glutathion sepharose 4B beads (Pharmacia ) are prepared by taking 1.33 ml of commercial available 75% slurry and spinning it at 500 x g in a 15 ml Falcon tube for 5 minutes. The supernatant is removed and 10 ml of cold PBS is added before mixing. The mixture is centrifuged at 500 x g in a Falcon tube for 5 minutes. The supernatant is removed and the pellet is re-suspended with 1 ml of cold PBS to give a 50 slurry.
  • Affinity purification of the target recombinant GST fusion protein can be carried out by the following methods:
  • Elute protein with free glutathione solution and the eluted fusion protein can be directly used for chicken immunization, or the GST portion can be cleaved from the fusion protein before the protein is used in animal immunization.
  • thrombin Sigma chemicals
  • Stock solution up at 1,000 cleavage unit per ml in PBS.
  • T he recombinant in the supernatant can be further separated on the SDS-polyacrylamide gel.
  • the eluted recombinant protein from SDS- PAGE gel or the directly sliced gel containing the recombinant protein can be used for the animal immunization.
  • Intein-mediated purification with an affinity chitin-binding tag is a novel protein purification system which utilizes the inducible self-cleavage activity of a protein splicing element, i.e., intein, to separate the target protein from the affinity tag (Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M-Q, Benner, J. (1998) Utilizing the C- terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109-5115.).
  • a protein splicing element i.e., intein
  • a target protein is fused to a tag containing the intein and the chitin binding domain, which allows affinity purification of the fusion precursor on a chitin column.
  • thiols such as DTT, b- mercaptoethanol or cysteine
  • the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag, resulting in a single-column purification of the target protein.
  • the commercially available system such as the IMPACT-CN system (New England Biolabs) contains expression vectors pTYB vectors, which allow fusion of the cleavable intein tag to either the C-terminus or N-terminus of the target protein. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein.
  • the same or compatible restriction sites are designed in the multiple cloning region of pTYB2 and pTYB12 vectors.
  • pTYBl and pTYBl 1 vectors allow the cloning of a target gene immediately adjacent to the intein cleavage site. This results in the purification of a native target protein without any vector- derived extra residues after the cleavage.
  • the pTYB vectors use a T7 promoter and the lac I gene to provide stringent control of the fusion gene expression. Binding of the lac repressor to the lac operator sequence immediately downstream of the T7 promoter suppresses basal expression of the fusion gene in the absence of IPTG induction.
  • the vectors also contain the origin of DNA replication from bacteriophage Ml 3, which allows the production of single-stranded DNA by helper phage, e.g., M13KO7 helper phage (NEB #N0315) superinfection of cells bearing the plasmid.
  • pTYB vectors carry the Ampr gene marker (the bla gene), which conveys ampicillin resistance to the host strain.
  • An affinity matrix is used for the isolation of the fusion precursor containing the target protein. Twenty ml of chitin beads ( ⁇ 50-100 ⁇ m in size) are supplied as a 38 ml slurry in 20% ethanol.
  • the binding capacity, which has been tested using the control vector pMYB5 is 2 mg of eluted MBP protein per ml of chitin beads.
  • CBD-based fusion protein expression and purification system Cellulose is an attractive matrix for affinity purification and immobilization mainly because of its combination of excellent physical properties and low price. Cellulose is commercially available in many different forms, such as cotton wool, filters, beads, powders, fibers, hydrogel, membranes, and sheets of defined porosity. To exploit the characteristics of this matrix, investigators have used a protein domain that naturally binds to cellulose: the cellulose binding domain (CBD).
  • CBD cellulose binding domain
  • CBDs provide a specific means for linking enzymes or other proteins on cellulose:
  • CBD-Protein- A for the purification of IgG
  • CBD-Streptavidin for different applications uses biotinylated molecules
  • CBD- Alkaline phosphatase can be used.
  • Cellulose binding domains are found in nature as discrete domains in proteins, such as in cellulases ( Gilkes, N.R., Warren, R.A.J., Miller, R.C., and Kilburn, D.G. (1988) J.
  • CBDs concentrate the catalytic domains on the surface of the insoluble cellulose substrate.
  • the CBD is part of a scaffoldin subunit that organizes the catalytic subunits into a cohesive multi-enzyme complex known as a cellulosome (Bayer, E., Morag, E., and Lamed, V.R. (1994) Trends Biotechnol. 12, 379- 386. ).
  • the cellulosome is responsible for efficient degradation of cellulosic substrates.
  • Cellulose is an unbranched homopolymer of ⁇ (l-4) linked glucose subunits.
  • Crystalline cellulose presents a surface array of parallel, closely-packed cellulose chains to a CBD.
  • Amorphous cellulose presents antiparallel or disordered chains to a CBD.
  • the binding sites of families I, II, and III CBDs are adapted to binding to a surface, and the family IV CBD to single molecules. Not surprisingly, only family IV binds to soluble cellulose derivatives and also to cello-oligosaccharides.
  • CBDs differ in both their binding and elution properties with different cellulose matrices. All of the CBDs that bind to crystalline cellulose and chitin (a homopolymer of ⁇ [l-4] linked n-acetyl-glucosamine) have very similar affinities with binding constants in the micromolar range. The family I CBDs bind reversibly, whereas the family II and III CBDs seem to bind irreversibly. Even with protracted washing, CBD fusion proteins derived from families II and III do not desorb from cellulose. The "irreversible" nature of this binding is not entirely understood; however, it may be related to the characteristics of the CBD-cellulose interactions and the properties of the cellulose itself.
  • Cellulose presents an array of multiple overlapping binding sites to CBDs. Also, binding is believed to be mediated by multiple reversible interactions between glucose molecules in the cellulose and amino acids in the CBD. Therefore, desorption would likely require the simultaneous breakage of multiple interactions without the reestablishment of those interactions on neighboring, overlapping binding sites. Although fusion of heterologous protein to a CBD has little effect on the affinity of the CBD for cellulose, it can affect desorption of the CBD from cellulose.
  • the CBD gene is linked to the gene of the protein of interest.
  • the resulting fusion protein binds strongly to cellulose and ideally retains the biological activity of the fusion partner. Binding is stable over a wide range of conditions (pH 2-10, high and low salt concentrations).
  • the fusion protein can be eluted from cellulose with distilled water in some cases (Ong, E., Gilkes, N.R., Warren, R.A.J., Miller, R.C., Jr., and Kilburn, D.G. (1989) Bio/Technol. 7, 604-607. Tomme, P., Gilkes, N.R., Miller, R.C., Jr., and Warren, R.A.J.
  • a chemical or protease cleavage site can be engineered between the CBD and the fusion partner enabling recovery of the target protein without the CBD.
  • CBDs derived from families II and III have been developed, such as pET and pBACTM vectors which use three CBD domains by Novagen, Inc. and is called CBD-TagTM sequences.
  • pET and pBACTM vectors which use three CBD domains by Novagen, Inc. and is called CBD-TagTM sequences.
  • Elastin-like polypeptides are oligomeric repeats of the pentapeptide Val-Pro-Gly-Xaa-Gly that undergo a reversible inverse temperature transition. They are highly soluble in water below the inverse transition temperature (Tt) but undergo a sharp (2-3°C) phase transition when the temperature is raised above Tt, leading to desolvation and aggregation of the polypeptide.
  • the target protein can be fused to ELP domain such as ELP 30 or ELP 60 to purify the recombinant protein (Dan E Meyer & Ashutosh Chilkoti ,. Purification of recombinant protein by fusion with thermally-responsive polypeptides. Nature Biotechnology . Voll7, pp: 1112-1115, 1999).
  • Advantages of this method, termed" inverse transition cycling" include technical simplicity, low cost ease of the scale-up, and capacity for the multiplexing.
  • the target protein can be easily purified by temperature-dependent centrifugation, and the purified protein can be used in the avian immunization directly.
  • the type and quality of the adjuvant used are of critical importance in determining the immune response, which should, ideally, be the induction of high serum and egg yolk antibody titers.
  • the use of an adjuvant, especially FCA can lead to a local tissue reaction at the injection site ( Wanke, R., Schmidt, P., Erhard, M.H., Sprick-Sanjose Messing, A.,
  • the expected antibody response can be generated by using an oil emulsion-type of adjuvant, such as Freund's incomplete adjuvant (FLA). No differences have been seen in the IgY response when FLA has been used for the primary immunization instead of FCA.
  • FLA Freund's incomplete adjuvant
  • Other types of adjuvant can also be used, such as Specol (Boersma, W.J.A., Bogaerts, W.J.C., Bianchi, A.I.J. & Claassen, E. (1992).
  • Adjuvant properties of stable water-in-oil emulsions evaluation of the experience with Specol. Research in Immunology 143: 503- 512; product no. 792500, ID-DLO, Lelystad, The Netherlands) and the lipopeptide, Pam3- Cys-Ser-(Lys)4.
  • the adjuvants A1PO4, Al(OH)3 and saponin have been found to induce only very low antibody responses.
  • it is important to first test the efficacy and quality of emulsion-type adjuvants according to standardized procedures (Herbert, W.J. (1967) Methods for the preparation of water-in-oil and multiple emulsions for use as antigen adjurants, and notes on their use in imminization procedures. In Handbook of Experimental Immunology (ed. D.M. Weir), pp. 1207-1214. Oxford: Blackwell).
  • vaccinate chickens that are at least 7 weeks of age, preferably at two injection sites, with volumes of about 0.5-1 ml. The total volume injected will affect the tissue reaction induced.
  • chickens kept under field conditions are vaccinated intramuscularly (i.m.) in the breast muscle.
  • chickens can also be vaccinated subcutaneously (s.c.) in the neck.
  • s.c. subcutaneously
  • Intramuscular injection in the leg should be avoided, since this could lead to lameness.
  • the total number of vaccinations required will depend upon the type and dose of the antigen, as well as on the particular adjuvant employed. In any case, at least two immunizations should be given. If the antibody titers begin to decrease, booster immunizations can be given during the laying period.
  • a primary vaccination and a booster should be given before the laying period, with an interval between these of about at least 6 weeks for emulsion-type adjuvants and about 4 weeks for lipopeptide adjuvants.
  • Yolk antibody titers should be checked 14 days after the last immunization; if the antibody titers are low, revaccination should be considered.
  • chickens can be used for the whole laying period, depending on the antibody titers induced. It is desirable to start with a group of chickens, and to select high responding animals which can then be kept for a longer period of time.
  • Adjuvant and volume Equal volume, most of time about 0.4 ml, of Freund's complete adjuvant, mixed with 100-200 ug antigen in 0.4 ml PBS buffer;
  • Injection site For young laboratory chickens, Intramuscular (field studies) and for older laboratory chickens, subcutaneous, both with an injection volume ⁇ 1 ml;
  • Injection frequency 2-3 times with boosters during laying period
  • the present invention provides an array of antibodies attached on a solid surface.
  • Any antibodies whether polyclonal, monoclonal, single chain, Fc fragment, Fab fragment, F(ab) 2 fragment, or a mixture thereof, can be used to produce the antibody arrays.
  • the array comprises antibodies that specifically bind substantially to proteins or peptides encoded by a set of pre-selected DNA sequences isolated from a biosample.
  • the set of pre-selected DNA sequences can be a cDNA library, such as a cDNA library isolated from animal, plant, fungus and bacterium cells.
  • the cDNA library is isolated from human cells.
  • the cDNA library can be a specialized cDNA library, such as a tissue or organ specific cDNA library, a developmentally-regulated cDNA library, or a cDNA library is isolated from physiologically normal or physiologically abnormal cells.
  • the antibodies used in the array are produced by the processes described in the above Sections A and B.
  • array shall be taken to mean any ordered arrangement of a plurality of specified integers, including both liner and non-linear arrangements of a plurality of antibodies or antibody variants or derivatives.
  • the array can be arranged on a grid, such as in microtitre wells, on a membrane support or silicon chip, or on a grid comprising a plurality of polymeric pins.
  • the array can be produced on any suitable solid surface, including silicon, plastic, glass, polymer, such as cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene, ceramic, photoresist or rubber surface.
  • silicon surface is a silicon dioxide or a silicon nitride surface.
  • the array is made in a chip format.
  • the solid surfaces may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other purous membrane, non-porous membrane, e.g., plastic, polymer, perspex, silicon, amongst others, a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilizing proteins and/or conducting an immunoassay.
  • PVDF polyvinylidene difluoride
  • the antibodies can be attached to the solid surface by any methods known in the art (see generally, WO 99/39210, WO 99/40434).
  • the antibodies can be attached directly or through linker(s) to the surface.
  • the antibodies can be attached to the surface through non-specific, specific, covalent, non-covalent, cleavable or non-cleavable linkage(s).
  • the cleavable linkage can be cleavable upon physical, chemical or enzymatic treatment.
  • the arrays can be arranged in any desired shapes such as linear, circular, etc.
  • antibody array can be printed on a solid surface using pins (passive pins, quill pins, and the like) or spotting with individual drops of solution (WO 99/40434).
  • Passive pins draw up enough sample to dispense a single spot.
  • Quill pins draw up enough liquid to dispense multiple spots.
  • Bubble printers use a loop to capture a small volume which is dispensed by pushing a rod through the loop.
  • Microdispensing uses a syringe mechanism to deliver multiple spots of a fixed volume.
  • solid supports can be arrayed using piezoelectric (ink jet) technology, which actively transfers samples to a solid support.
  • the methods disclosed in WO 95/35505 can also be used. The method and apparatus described in WO 95/35505 can create an array of up to six hundred spots per square centimeter on a glass slide using a volume of 0.01 to 100 nl per spot.
  • Suitable concentrations of antibody range from about 1 ng/ ⁇ l to about 1 ⁇ g/ ⁇ l.
  • other methods of creating arrays including photolithographic printing (Pease, et al, PNAS 91(ll):5022-5026, 1994) and in situ synthesis can be used.
  • BSA bovine serum albumin
  • a method for assessing proteomics profile of a biosample comprises: 1) dividing a plurality of antibodies into an unlabelled portion and a labeled portion; 2) attaching the unlabelled antibodies on a solid surface to form an array of unlabelled antibodies on said solid surface; 3) contacting said array of unlabelled antibodies formed in step 2) with a biosample to retain antigens contained in said biosample that specifically bind to said unlabelled antibodies; and 4) detecting said retained antigens by contacting, said retained antigens with said labeled antibodies, thereby proteomics profile of said biosample is assessed.
  • the plurality of antibodies used in the above methods are produced and characterized against a plurality of antigens encoded by a set of pre-selected DNA sequences isolated from a bio-sample via a process comprising the steps: 1) cloning each of said DNA sequences into a dual-expression vector that is capable of expressing said DNA sequences in avian cells, non-avian cells or in vitro expression systems; 2) delivering said DNA sequence in said dual-expression vector formed in step 1), or mRNA or protein encoded by said DNA sequence, or a mixture thereof, to an avian species in an amount sufficient to induce detectable production of antibodies to an antigen encoded by said DNA sequence, and recovering said antibodies from said avian species; 3) delivering said DNA sequence, or mRNA encoded by said DNA sequence, or a mixture thereof, which is delivered to said avian species in step 2), to said non-avian cells, and recovering proteins or peptides encoded by said DNA sequence from said non-avian cells, or expressing and recovering proteins
  • a method for identifying physiologically distinguishable markers associated with a physiologically abnormal bio-sample comprises: 1) assessing proteomics profile of said physiologically abnormal bio-sample through the above-described method; 2) assessing proteomics profile of a comparable physiologically normal bio-sample through the above-described method; and 3) comparing the proteomics profile obtained in step 1) with the proteomics profile obtained in step 2) to identify physiologically distinguishable markers associated with a physiologically abnormal biosample.
  • the physiologically abnormal bio-sample is isolated from an organism, preferably mammals or humans with a disease or disorder, and the method is used in prognosis, diagnosis, or monitoring treatment of such diseases or disorders.
  • the exemplary the diseases or disorders that can be monitored by the present methods include cancers, immune system diseases or disorders, metabolism diseases or disorders, muscle and bone diseases or disorders, nervous system diseases or disorders, signal diseases or disorders, or transporter diseases or disorders.
  • a method for identifying a substance that modulates proteomics profile of a biosample comprises: 1) assessing proteomics profile of a bio-sample through the above-described method in the presence of a test substance; 2) assessing proteomics profile of said bio-sample through the above-described method in the absence of said test substance; and 3) comparing the proteomics profile obtained in step 1) with the proteomics profile obtained in step 2), whereby the proteomics profile obtained in step 1) is different from the proteomics profile obtained in step 2) identifies the test substance as a modulator of said proteomics profile of said bio-sample.
  • the method can be used in screening a single test substance at a time, the method is preferably used in a high-throughput format, i.e., a plurality of test substances are tested simultaneously.
  • test substance refers to a chemically defined compound (e.g., organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins, etc.) or mixtures of compounds (e.g., a library of test compounds, natural extracts or culture supematants, etc.) whose effect on the promoter to be analyzed is determined by the disclosed and/or claimed methods herein. Any substances can be screened using the present screening methods for finding drug candidates for modulating proteomics profile of a biosample.
  • a chemically defined compound e.g., organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins, etc.
  • mixtures of compounds e
  • a combinatorial library is used in the screening assays.
  • Methods for synthesizing combinatorial libraries and characteristics of such combinatorial libraries are known in the art (See generally, Combinatorial Libraries: Synthesis, Screening and Application Potential (Cortese Ed.) Walter de Gruyter, Inc., 1995; Tietze and Lieb, Curr. Opin. Chem. Biol, 20):363-71 (1998); Lam, AnticancerDrugDes., 12(31:145-67 (1997); Blaney and Martin, Curr. Opin. Chem. Biol, 1(1): 54-9 (1997); and Schultz and Schultz, Biotechnol. Prog., 12 ⁇ 6):729-43 (1996)).
  • the above described processes, methods and antibody arrays can be used for identifying physiologically distinguishable markers associated with a physiologically abnormal bio-sample, or for identifying substances that modulate proteomics profile of a biosample.
  • the present invention further encompasses integrated databases for identification of genes and proteins (IDIGAP).
  • IDIGAP integrated databases for identification of genes and proteins
  • the basic concept of IDIGAP is a development of the AMIGAP technology.
  • the key point is the establishment of an integrated-database system that includes, but is not limited to, genomic, cDNA, expression vector, recombinant protein, and antibody databases, which integrated-database system forms an bioimformatic network for identification of genes and proteins.
  • the method for producing such an integrated-database system uses immunization of chicken or other avian species with nucleic acid or recombinant proteins as immunogens to generate avian antibodies, e.g., IgYs, with a broad recognition spectrum in a high-throughput format.
  • Figure 14 summarizes the inter-relationship of the five databases and its usage in identification of the target protein and related gene.
  • Figure 15 illustrates a specific process of application of IDIGAP for identifying a disease-related protein and gene.
  • mRNAs can be isolated from one or more tissues of interest that are normal or abnormal.
  • cDNA libraries can be made from the mRNA to form cDNA databases.
  • the cDNA can be sequenced and numbered with identities, e.g., A, B, C, n.
  • the sequence information of each cDNA can be used for constructing genomic databases with the numbering of each gene or sequence corresponding to the cDNA database.
  • the cDNA can also be cloned in a dual-expression vector system to form a vector database, again with the corresponding numbering.
  • the vectors can be, on one hand, used for generating recombinant proteins through expression system such as bacterial, yeast, baculovirus system, and on the other hand, used for immunization of chicken or other avian species. These two processes can generate purified recombinant proteins and avian antibodies, e.g., IgYs, that can form two separate databases with the corresponding numbering as the vector database.
  • an inter-related databases network is established. By using the antibodies to screen tissue slides, disease related proteins can be identified. The related genes can then be identified through the database network.
  • the present invention encompasses an integrated database for identification of genes and proteins, which integrated database comprises a genomic sequence subdatabase, a cDNA sequence subdatabase, a dual expression vector subdatabase which provides information for a plurality of vectors that are capable of directing expression in an avian species and in a non-avian species or an in vitro expression system, a protein sequence subdatabase, an antibody subdatabase and means for linking information in one subdatabase to information in other subdatabases, wherein said genomic DNA sequences, cDNA sequences, dual expression vectors, proteins or peptides and avian antibodies correspond to each other according to the central dogma and antigen-antibody binding specificity.
  • the dual expression vector directs expression in an avian species and in a non-avian species such as a bacterium, a yeast, an insect, or a mammal.
  • a non-avian species such as a bacterium, a yeast, an insect, or a mammal.
  • the antibody subdatabase provides information for a plurality of IgY antibodies produced in the avian species.
  • genomic sequence database including the genomic DNA or genomic DNA
  • RNA sequence database cDNA sequence database
  • dual expression vector database which provides information for a plurality of vectors that are capable of directing expression in an avian species and in a non-avian species or an in vitro expression system
  • protein sequence database and antibody database can be used in the integrated database.
  • the publicly accessible genomic sequence database, cDNA sequence database and protein sequence database including the ones listed at http:Wwww.ncbi.nlm.nih.gov, can be used.
  • the genomic sequence subdatabase, the cDNA sequence subdatabase, the dual expression vector subdatabase which provides information for a plurality of vectors that are capable of directing expression in an avian species and in a non-avian species or an in vitro expression system, the protein sequence subdatabase, and the antibody subdatabase can be constructed by any methods known in the art or any methods described in this Application.
  • the dual expression vector subdatabase which provides information for a plurality of vectors that are capable of directing expression in an avian species and in a non-avian species or an in vitro expression system and the protein sequence subdatabase can be constructed based on bioimformatic information identified by using the methods described in the above Sections C and D.
  • bioimformatic information can be delivered or imported into known database format such as Excel, Lotus, Access, DB2, SQL Sever and Oracle, etc.
  • the imported information can then be characterized or manipulated within each database using routine procedures or softwares, many of which are built-in elements of the databases.
  • Means can also be provided for linking, e.g., importing, exporting, indexing or synchronizing, information among different databases.
  • SQL Net or Net 8 software can be used for such purposes.
  • many databases have internal structures or languages for enabling or facilitating such cross- database exchanges.
  • the present invention further encompasses a method for generating an integrated library for identification of genes and proteins, which method comprises: 1) selecting and marking a plurality of DNA sequences from a genomic library; 2) selecting and marking a plurality of cDNA sequences from a cDNA library that correspond to said selected and marked plurality of genomic DNA sequences; 3) cloning said plurality of selected and marked cDNA sequences into a dual expression vector that is capable of directing expression of said plurality of selected and marked cDNA sequences in an avian species and in a non-avian species or an in vitro expression system; 4) producing a plurality of proteins or peptides encoded by said plurality of selected and marked cDNA sequences by delivering and expressing said dual vector containing said plurality of selected and marked cDNA sequences into said non-avian species or said in vitro expression system; and 5) generating antibodies from an avian species using said dual vector formed in step 3) via nucleic acid vaccination or using proteins or peptides
  • the method further comprises a step of conducting immunoreactions between said antibodies generated in step 5) with said proteins or peptides generated in step 4) to characterize the immunospecificity of said antibodies.
  • the method further comprises a step of conducting immunoreactions between said characterized antibodies with a biosamples from which genomic library is isolated to determine the proteomics profile of the selected and marked plurality of genomic DNA sequences.
  • the present invention further encompasses a method for generating an integrated database for identification of genes and proteins, which method comprises: 1) delivering bioimformatic information of the plurality of genomic DNA sequences, the plurality of cDNA sequences, the plurality of dual expression vectors, the plurality of proteins or peptides, and the plurality of avian antibodies obtained using the above-described methods into the corresponding genomic DNA, cDNA, dual expression vector, protein or peptide, and the avian antibody subdatabases; and 2) providing means for connecting the bioimformatic information from one subdatabase to any or all of the other subdatabases.
  • the method further comprises a step of delivering bioimformatic information of the immunospecificity of the avian antibodies obtained using the methods described in the above Section C into the integrated database.
  • the method further comprises a step of delivering bioimformatic information of the proteomics profile of the selected and marked plurality of genomic DNA sequences, which can be obtained according to the methods described in the above Section C, into the integrated database.
  • pDual vector purchased from STRATAGEM- Inc. (La Jolla, CA), was used as template vector for amplification of both CMV/T7 promoter expression cassette.
  • 10 ngpDual plasmid DNA was heated to 95°C for 5 minutes and then mixed with 10 pM of both CMV-1 (5'CACCCTGAATTGACTCTCTTTC3') (SEQ ID NO:l) and PacII-1 (5 ⁇ TATGAATTCTTAATTAAGATCTCCATGGTGGCCTCTCCTTC3') (SEQ ID NO:2) oligos using standard PCR reaction (Promega PCR Kit).
  • PCR reaction was performed as follow: 40 cycles at 95°C for 1.45 minutes, 55°C for 1.30 minutes, 72°C for 2 minutes. Finally, the PCR product was further incubated at 72°C for 10 minutes.
  • the Products was named as Fragment I (0.75 kb).
  • 10 ng pDual plasmid DNA was amplified by PCR using the above-described condition with oligo Pad (5'CGCGGAATTCGCGGCCGCTACCAGGTAAGTGTACC3') (SEQ ID NO:3) and oligo ter-2 (5'CGAGTAGTTTAAACAAAAAACCCCTCAAGTCCCG3') (SEQ ID NO:4).
  • the Product was named as Fragment-II (0.6kb).
  • the purified pT7Blu(R) plasmid purchased from Novagen Inc., was used as DNA template for PCR amplification.
  • 10 ng pT7Blu(R) plasmid in 100 ul PCR reaction mixture, two oligos T7A and T7B were added into reaction and PCR was performed using the same condition as described above.
  • T7A 5 ⁇ GATCTGTTTAAACCAGGTGGCACTTTTCGG3' (SEQ ID NO:5)
  • T7B 5 ⁇ GATCTGTTTAAACAGCTGTTTCCTGTGTGA3' (SEQ ID NO:6).
  • the 2.1 kb PCR product was then digested with BgLII and gel-purified for self-ligation.
  • the resulting vector was named pT7* which carries two unique Pmel sites.
  • Linker- 1 (5'AACCCTCTTCCATGAGCCCACTCGTCTCCTCCCTCCTGCTCCTGGCCGCCCTG CCAGGGCTGATGGCGGCC3') (SEQ IDNO:8), Linker-2:
  • Linker- 1 and Linker-2 oligo were mixed in 50 ul PCR reaction condition without Taq DNA polymerase, heated to 94°C for 5 minutes, and then was slowly cooled to room temperature. After adding 2.5 units of Taq DNA polymerase, the PCR reaction was performed using the same condition as above but was only run for 10 cycles.
  • the 105 bp PCR product was digested with Eaml 104-1 and inserted into pDual vector's Eaml 140-1 site. The resulting vector was named pDual-S.
  • the 1.5 kb DNA fragment which carries the gene expression cassette was generated by further PCR amplification with the CMV-1 and Ter-2 oligos. After Pmel digestion, the 1.5 kb DNA fragment was inserted into pT7* vector's Pmel site to generate the dual function vector pS&DV-S vector.
  • ThO T cell Stimulation of ThO T cell with LL-4 leads to the development of Th2 CD4 " helper T cell, which will secrete cytokine to promoter B cell development, including IL-4, 11-5, 11-6, and IL-10.
  • Stimulation of ThO cells with the proinflammatory cytokine IL-12 and IFN- ⁇ leads to development of the Thl CD4 + helper T cell. These cells secrete cytokines that will promoter the development of CD8 + cytotoxic T lymphocytes (Koprowski et al DNA vaccination genetic vaccination. 1998. Springer- verlag Berlin heidelberg.).
  • the DNA was delivered into the target site (chicken back skin) using a handheld, helium-driven ballistic gene gun with equivalent of 200 ng plasmid (Sanford, et al., Technique, 1991, 3:3-16). The pressure in the gun was adjusted to 1200 psi. After DNA injection, at different post-injection day, the eggs from the immunized chickens were collected and stored at 4°C and IgY was purified using the protocol described in ⁇ 6.8. In this experiment, repeated DNA injection with same amount of plasmid DNA was performed to observe the host immuno-response for antigen.
  • the RNase H domain of the HBV polymerase protein was PCR amplified using Pol-
  • the constructed plasmids were transformed into BL21(DE3) competent cells, clones were transferred into 3 ml LB medium which contains carbenicillin (Sigma Inc. cat
  • the purified protein was eluted from the column with 10 ml IX strip buffer, which contains 6 M urea.
  • the purified protein sample was transferred into a dialysis tube and was dialyzed against PBS overnight at 4°C in 4 Liter volume. Finally, the protein sample in the dialysis tube was further concentrated into 1 ml volume by Amicon spin concentration column (Amicon Inc, MW cut of: 10,000 dalton). After checking protein concentration, 3 ul of purified protein were separated on 12.5% SDS-PAGE. The purity of the recombinant protein, measured by the density of protein bands using molecular densitometry (Molecular Dynamic Inc), is more than 93%.
  • each of different bars represents different chickens.
  • chicken was immunized with pCMV-HBx vector; in Figure 5B, chicken was immunized with pCMV-HBV-pol vector; and in Figure 5C, chicken was immunized with pZeoSV2- hCD34.
  • Assay time point is as the following: Preimmune (Pre); or 12 days after boost 1 (Bl), Boost 2 (B2) and boost 3 (B3). Analysis of the multiple DNA vaccination host immuno-reaction data shows that 12 days after single DNA injection, chicken specific antibody production already reached the detectable level.
  • hepatitis B X gene expression vector was performed as the following. Ten ng of pTKHH2 DNA (HBV full-length viral genome dimer plasmid) was mixed with MF18 (MF18: 5'GGAAGCTTGCCGCCATGGCTGCTAGGCTGTGC3') (SEQ ID NO: 10) and MF19 (MF19: 5'GTGGAGACGGATTAGTACCATGGCC3') (SEQ LD NO:l 1) oligo in 100 ul PCR reaction tube. HBV polymerase gene was PCR amplified in the following condition: at 95°C for 1.30 minutes, at 55°C 1.30 minutes and at 72°C for 2 minutes; for a total of 40 cycles.
  • PCR product was incubated at 72°C for 10 minutes.
  • the 488 bp PCR product was gel purified and then digested with Hindlll and Kpnl.
  • the digested HBx fragment was inserted into mammalian expression vector pTTW- 1 vector (Condreay et al., J. Virology, 1990, 64:3249-3258), which was digested with same enzymes to generate the pCMV-HBx plasmid.
  • the expression of the HBx protein was tested by transfecting the pCMV-HBx into human hepatocellular carcinoma cell line HepG2 cells.
  • 1 x 10 6 HepG2 cells were seeded in 10 cm culture dish in 10 ml DMEM medium which was supplemented with 10% fetal calf serum at 37°C in CO 2 incubator overnight.
  • 10 ml fresh pre-warmed DMEM medium supplemented with 10% fetal calf serum was replaced.
  • Five ug purified pCMV-HBx plasmid were mixed in calcium precipitation mixture according the manufacturer's protocol (Promega Inc.'s calcium transfection kit). After the mixture precipitated at room temperature for 30 minutes, the mixture was slowly dropped into HepG2 cells and cultured for 12 hours. Next day, 10 ml pre-warmed fresh medium was replaced and the cells were cultured for one more day.
  • Transfected cells were washed with 10 ml cold PBS buffer and cells were collected using rubber policeman in 1.5 ml PBS. After centrifugation, the cell pellet was resuspended in 100 ul H 2 O, and 20 ul cell sample was mixed with same volume SDS- PAGE loading buffer and boiled for 3 minutes. The boiled sample was subjected to the max-speed Eppendorf centrifugation for 2 minutes and 5 ul of the supernatant were loaded on 12.5 % gel for SDS-PAGE separation. The extracted cellular protein was demonstrated to be positive for HBxAg expression using Western Blot assay with specific rabbit anti- HBx antibody (1: 800 dilution) (Wu et al., Cell, 1990, 63:687-695).
  • Hepatitis B polymerase antigen specific expression vector Construction of the hepatitis B polymerase (HBV pol) gene expression vector was performed as following. In 100 ul PCR reaction as described in ⁇ 6.4, pTKHH2 plasmid
  • DNA template was mixed with oligos MF26
  • HBVpol-2 (5 ⁇ AGAGCTCGCCACCATGGCCCTATCCTATCAAC3') (SEQ ID NO:12) and HBVpol-2 (5'TCACCTTAAGGTGTTGGAAGGTGGTTTGA3') (SEQ ID NO: 13).
  • the 868 bp HBV pol 5' end DNA fragment was gel purified and digested with Sail and EcoRI, then inserted into vector pGEM3Z (Promega Inc.) which was digested with the same enzymes to generate the plasmid pGEM3Zpol-5'.
  • pTKHH2 Vector another 1638 bp 3 'end of HBV polymerase DNA fragment was PCR amplified using pTKHH2 plasmid mixed with the following oligos, Pol-3
  • the PCR product was digested with EcoRI and Hindlll.
  • the digested fragment was inserted into pGEM3Zpol-5'EcoRI-HindIII site to generate the 2874 bp full-length HBV polymerase gene.
  • the resulting plasmid was named p3Zpol.
  • the HBV polymerase gene was digested with Sad first and then filled in with Klenow reaction (Sambrook et al., Molecular Cloning, Second Edition, Plainview, N.Y.
  • HBV polymerase protein was also tested by transfecting the pCI- HBV-pol into human hepatoma cell line HepG2 as described in ⁇ 6.4.
  • the extracted cellular protein was demonstrated to be positive using Western Blot assay with specific rabbit anti-HBV polymerase peptide antibody (Feitelson et al., Clinics In Laboratory Medicine, 1996, W.B.Saunders Com).
  • CD34 positive cell line KG-la was used for RNA extraction (Simmons et al., J. Immunol, 1992,
  • 2X10 6 cultured KG-la cells were suspended in 5 ml buffer (4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol, pH 7.0).
  • the following reagents were added, punctuated by vortexing of the tube: 2 M sodium Acetate pH 4.0 (0.5 ml), Phenol (5 ml), and chloroform (1 ml).
  • the tubes were centrifuged at 10,000 g (7,000 rpm) for 10 min. Isopropanol (5 ml) was added to the upper phase and incubated on ice for 10 min, followed by centrifugation as describe above.
  • RNA pellet was dissolved in 1 ml 4 M LiCL and transferred to a microcentrifuge tube. The original tube was rinsed with 0.5 ml LiCl and the pellet was vortexed for 5 min in the combined liquid. RNA was pelleted by centrifugation (10 min), resuspended in 1 ml 4M LiCl and pelleted again. The pellet was thoroughly resuspended in TE/0.5% SDS and extracted with an equal volume of chloroform/isoamyl alcohol (24:1). The aqueous phase was extracted a second time before precipitation of RNA by adding 2 M sodium acetate (0.1 ml) and isopropanol (600 ul). RNA was pelleted and resuspended in water.
  • RNA was used for reverse transcription (RT) reaction with oligo dT 18 primer, following the manufacturer's protocol (BRL Life Science Inc., RT kit).
  • Full-length human CD34 cDNA was PCR amplified using oligo hCD34-l (5'GAAGGATGCTGGTCCGCAGGGG3') (SEQ D NO:16) and hCD34-2 (5'CACCTAGCCGAGTCACAATTCG3') (SEQ ID NO:17) primers.
  • the PCR reaction was performed at the following condition: at 95°C for 1.30 minutes, at 53°C for 1.30 minutes, at 72°C for 2 minutes; and for a total of 40 cycles.
  • PCR product was incubated at 72°C for 10 minutes.
  • the 1.2 kb PCR product was directly inserted into the Hindi digested pUCl 8 vector (Phamacia Inc).
  • the resulting plasmid pUCl 8-hCD34 was confirmed to contain the full-length hCD34 sequence by DNA sequencing analysis using ABI 373 DNA sequencer and M13 primers.
  • the 1.2 Kb hCD34 fragment was gel purified and inserted into mammalian expression vector pZeoSV2+ (Lnvitrogen Inc.), via the Hindlll and EcoRI sites to generate the vector pZeoSV2-hCD34.
  • the expression of the pZeoSV2-hCD34 was confirmed by transfecting it into HeLa cells and immunostaining with mouse anti-human CD34 monoclonal antibody (Pharmingen Inc., CA).
  • Enzyme-linked immunoassay of chicken antibody to HbxAg a this experiment, purified E. coli derived HBxAg antigen was used for assaying chicken anti-HBx antibody which was generated from the DNA vaccination as described in
  • the protein precipitate formed was pelleted by centrifugation at 13,000 g for 10 min.
  • the supernatant was decanted and filtered through cheesecloth and PEG 6000 was added to bring the final concentration to 12%.
  • the mixture was stirred thoroughly and centrifuged again at 13,000 g for 10 min.
  • the pellet was redissolved to the original yolk volume in
  • IgY was purified on DEAE-cellulose by adsorption at
  • cob-derived recombinant HbxAg antigen (as described ⁇ 6.3) were separated on a 12.5% SDS-polyacrylamide gel and the separated proteins were transferred onto a PVDA membrane according to the manufacturer's protocol (Bio-Rad mini-gel kit).
  • Yolk antibodies purified by PEG 6000 precipitation as described in ⁇ 6.8, were diluted 1:1200 in Tris-Buffer saline, and 10 ml purified IgY solution were applied to the PVDA membrane and incubated at 37°C for 2 hours.
  • human B cells can be immortalized by EBV infection and mouse B cells can be immortalized directly with transfection of oncogenes, such as mutant p53 and Ras oncogenes.
  • Chicken B cells are selected for immortalization with chicken specific oncogene(s) using refroviral vectors transduction system, especially the lantiviral vector system which has the ability to infect the quiescence cells.
  • the ASV (Avian Sarcoma Virus) based vector has been widely used in transforming chicken cells (Kaplitt et al., Viral Vectors, Academic Press, 1995). This section describes the construction of an HIV-based vector containing chicken mutant p53 or Ras gene fragment, which can be used for chicken B cell immobilization.
  • HIV-1 based lantiviral vector (Naldini et al.,
  • Chicken mutant p53 oncogene is PCR amplified using following two oligos: Cp53-1
  • CITE DNA fragment is PCR amplified from pCITE-5b(+) plasmid (Novagen Inc.; Parks et al., J Virol, 1986, 60:376-384) and inserted into pT7-p53 vector in the downstream of p53 oncogene to produce the pT7-p53- CITE vector.
  • C-Ras-1 (5 ⁇ TGACCGAGTACAAGCTG3') (SEQ ID NO:20) and C-Ras-2 (5'TCACGATATCACGCATTTACAG 3') (SEQ ID NO:21)
  • the chicken Ras oncogene is amplified by PCR and the Ras oncogene DNA fragment is inserted into pT7-p53-CITE to generate the dual oncogene expression vector: pT7-p53- CITE-Ras.
  • LacZ gene DNA fragment in HIV-1 vector is replaced by chicken mutant p53-CITE-Ras oncogene DNA fragments to generate expression vector pHIV-l-Ch-p53-Ras.
  • the oncogenes' expression is driven under a single CMV promoter.
  • HIV-1 virus host cell specificity problem was overcome by pseudotyping with the G protein of vesicular stomatitis virus (VSV-G).
  • VSV-G vesicular stomatitis virus
  • the experiments to generate the HIV-1 based lantiviral vector transfection stock was performed as the following. Five ug pCMV- VSV-G plasmid, 5 ug HIV-1 help plasmid pCMV*R9 and 10 expression vector pHTV-lacZ were mixed and transfected into lxl 0 6 293 cells using calcium precipitation procedure (Promega Inc.).
  • the transduction of lantiviral vector for chicken cells was tested as the following.
  • transduced cells were fixed with 0.25% (v/v in PBS) glutaraldehyde solution for 15 minutes, and stained with X-Gal solution (1 mg/ml X- Gal, 2 mM MgCl 2 , 5 mM K_ 4 Fe(CN) 6 -3X H 2 O, 5 mM K 3 Fe(CN) 6 ) for 2 hours at 37°C.
  • X-Gal solution (1 mg/ml X- Gal, 2 mM MgCl 2 , 5 mM K_ 4 Fe(CN) 6 -3X H 2 O, 5 mM K 3 Fe(CN) 6 ) for 2 hours at 37°C.
  • the pseudotyped lantiviral stock title in this experiment was determined to be 1.32 X 10 5 /ml.
  • Spleen B cells of DNA vaccinated chickens are immortalized as the following.
  • Chicken spleen cells are collected and purified using Hypaque-Density Ficoll Gradient procedure (Sigma Inc.). After washing three times with PBS buffer, 1X10 5 mixed B cells are seeded in a six well plate in 1 ml MEM- 10% FCS medium and directly mixed with 2 ml viral stock solution overnight.
  • the viral stock supernatant is treated with 5 mM dNTP and 2 mM spermidine at 37°C for 2 hours to enhance the viral infectivity (Zhang et al., J. Virology, 1995, 69:3929-3932).
  • B cells Four ug/ml polybrene (Sigma Inc) is also added into B cell culture to enhance the viral transduction efficiency during the viral/cell incubation. After B cells are incubated with the viral solution at 37°C overnight, B cells are diluted into the single well culture (10 cells/well) which contains feeding cells in the 96 well plate (400-500 chicken B cells/well irradiated with 20 Gays). The transformed cells are incubated for two to three weeks, the grown cell supernatant are first tested for the production of IgY antibody (Davis, Ed., Methods in Molecular Biology, Monoclonal Antibody Protocols, 1995, Human Press), or screened for specific antigen binding IgY using ELISA as described above.
  • IgY antibody Daavis, Ed., Methods in Molecular Biology, Monoclonal Antibody Protocols, 1995, Human Press
  • each of the cDNA library 300 individual clones were picked up and cultured in 3 ml LB with shaking overnight.
  • Each plasmid was prepared using Quigen Tip20 kit and 1 ug plasmid DNA was sequenced using ABI377 automatic DNA sequencing system with primer suggested by the library manufacturer.
  • each host B cell After stimulated by a specific antigen, each host B cell generates a specific antibody, which either binds to antigen specific sequential domain or conformational structure domain.
  • a polyclonal antibody binds to a specific antigen through multiple binding sites.
  • the antibody-chip comprising groups of specific antibodies on solid matrix support can be used to capture the free target protein (antigens) in a protein sample solution. After washing steps, the same group of antibody which is conjugated with an enzyme, such as HRP, or a detectable marker such as fluorescence dye (FITC or C3) can be used to further bind those captured antigen because of multiple binding domains of polyclonal antibodies, and to determine binding signal density with substrate of the enzyme such as ECL system or laser emission system (flowcytometer).
  • AMIGAP of the present invention one can generate multiple antibodies to unknown proteins or functionally undefined proteins. After purification of each IgY antibody, one can divide each of the antibody into two fractions and label one of the fraction with biotin (PIERCE Inc. IL. EZ-Link Biotinylating Reagents). Hundreds or thousands specific unlabeled antibodies are individually and randomly spotted on two identical solid support matrix (e.g., 1 ug of each of antibody per spot on PVDF membrane or marked individual glass bead; and each spot or bead represents one known antibody). The spotted matrix is blocked with 5% BSA-PBS buffer to reduce the non-specific binding background.
  • biotin PIERCE Inc. IL. EZ-Link Biotinylating Reagents
  • Specific group of antibodies such as the antibodies targeting cell-cycle specific regulatory proteins or G-coupled receptor family proteins, can be used.
  • the antibody-chip can be air-dried and stored at 4°C in the sealed plastic bag for several months. Before performing the experiments, the antibody-chip can be activated by wetting the chip in PBS buffer for 30 minutes.
  • the system described here can be used in comparing. the target protein expression in two samples, such as liver tumor cells vis-a-vis normal liver cells or human lung cancer cells treated with anti-cancer drug vis-a-vis untreated control cells.
  • two target cell samples or tissues can be lysed by gentle detergents in PBS solution or freeze and thaw method (Sambrook et al., Molecular Cloning, Second Edition. Plainview, N.Y. Cold Spring Harbor Press, 1988). Same number of cells can be used for protein expression comparison.
  • cell lysate sample are measured with its protein concentration first and then equal amount of protein sample are loaded onto the antibody-chips. Usually, 1X10 6 cells per lysate sample or 50-100 ug proteins are used for each assay.
  • biotin labeled antibodies As cellular target proteins are captured by membrane-bound docking antibodies, functioning as sandwich fashion, those captured target proteins are detected by mixture solution of biotin labeled antibodies which corresponds to each of the originally spotted antibodies and incubated 37°C for 2 hours.
  • the signal density of the captured biotin- labeled antibodies is associated with the level of docking cellular protein level (antigen). Further quantification of captured biotin-labeled antibodies shows the antigen expression level in this assay. After washing 5 times in PBS buffer, the non-captured biotin-labeled antibodies on the antibody-chips are removed. Finally, avidin conjugated HRP (Sigma Inc. 1:8000 dilution in PBS with 2% BSA) is added and incubated for 15 minuets. After washing the antibody-chips with PBS solution for six times and soaking the antibody-chip with ECL substrate solution (PLERCE Inc. IL), the chip is exposed to X-ray film.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medical Informatics (AREA)
  • Evolutionary Biology (AREA)
  • Theoretical Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Databases & Information Systems (AREA)
  • Analytical Chemistry (AREA)
  • Bioethics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Communicable Diseases (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de production d'anticorps polyclonaux et monoclonaux dirigés contre un antigène chez une espèce aviaire, de préférence chez un poulet, au moyen d'une vaccination polynucléotidique. L'invention concerne également des procédés de détermination du profil protéomique d'un ensemble de séquences d'ADN présélectionnées isolées d'un échantillon biologique, de préférence le profil protéomique d'une banque d'ADNc humains. L'invention concerne en outre des procédés d'identification de marqueurs physiologiquement différenciables associés à un échantillon biologique physiologiquement anormal. L'invention concerne finalement des réseaux d'anticorps, des bases de données intégrées destinées à l'identification de gènes et de protéines, des vecteurs d'expression de gènes multi-fonctionnels, ainsi que des procédés de production et d'utilisation de ces réseaux d'anticorps, des bases de données intégrées et des vecteurs d'expression de gènes multi-fonctionnels.
PCT/US2001/014817 2000-05-16 2001-05-07 Procedes et vecteurs destines a generer des anticorps dans des especes aviaires et utilisations Ceased WO2001088162A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001259631A AU2001259631A1 (en) 2000-05-16 2001-05-07 Methods and vectors for generating antibodies in avian species and uses therefor

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US20489900P 2000-05-16 2000-05-16
US09/573,442 US6951742B1 (en) 1998-11-16 2000-05-16 Methods and vectors for generating antibodies in avian species and uses therefor
US60/204,899 2000-05-16
US09/573,442 2000-05-16

Publications (2)

Publication Number Publication Date
WO2001088162A2 true WO2001088162A2 (fr) 2001-11-22
WO2001088162A3 WO2001088162A3 (fr) 2002-09-19

Family

ID=26899888

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/014817 Ceased WO2001088162A2 (fr) 2000-05-16 2001-05-07 Procedes et vecteurs destines a generer des anticorps dans des especes aviaires et utilisations

Country Status (2)

Country Link
AU (1) AU2001259631A1 (fr)
WO (1) WO2001088162A2 (fr)

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003054021A3 (fr) * 2001-12-20 2004-02-12 Agricultural Research Council Bibliotheque combinatoire de fragments d'anticorps semi-synthetiques
CN1818651B (zh) * 2006-03-13 2010-07-21 东北农业大学 兔抗鹅IgY+IgY(△Fc)(H+L)辣根过氧化物酶标记抗体
WO2010107825A2 (fr) 2009-03-16 2010-09-23 Pangu Biopharma Limited Compositions et procedes comprenant des variants d'epissage d'histidyl-arnt synthetase presentant des activites biologiques non canoniques
WO2010120509A2 (fr) 2009-03-31 2010-10-21 Atyr Pharma, Inc. Compositions et procédés impliquant des aspartyl-arnt synthétases présentant des activités biologiques non canoniques
WO2011135459A2 (fr) 2010-04-29 2011-11-03 Medical Prognosis Institute A/S Méthodes et dispositifs permettant de prédire l'efficacité d'un traitement
WO2011140132A2 (fr) 2010-05-03 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de phénylalanyl-alpha-arnt-synthétases
WO2011139714A2 (fr) 2010-04-26 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps se rapportant à des fragments protéiques de la cystéinyl-arnt synthétase
WO2011139986A2 (fr) 2010-05-03 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps liées à des fragments protéiques d'arginyle-arnt synthétases
WO2011139853A2 (fr) 2010-04-28 2011-11-10 Atyr Pharma, Inc. Compositions thérapeutiques, diagnostiques et d'anticorps à base de fragments de protéines d'aminoacyl-arnt synthétases
WO2011139799A2 (fr) 2010-04-27 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps se rapportant à des fragments protéiques d'isoleucyl arnt synthétases
WO2011139854A2 (fr) 2010-04-29 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à based'anticorps associées à des fragments protéiques d'asparaginyl-arnt-synthétases
WO2011140135A2 (fr) 2010-05-03 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées des fragments protéiques de méthionyl-arnt-synthétases
WO2011139907A2 (fr) 2010-04-29 2011-11-10 Atyr Pharma, Inc. Découverte innovatrice de compositions thérapeutiques, diagnostiques, et d'anticorps associées aux fragments protéiques des valyle arnt synthésases
WO2011140267A2 (fr) 2010-05-04 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de complexe multi-arnt synthétase p38
WO2011143482A2 (fr) 2010-05-14 2011-11-17 Atyr Pharma, Inc. Découverte de compositions inédites de nature thérapeutique, diagnostique et à base d'anticorps contenant des fragments protéiques de phénylalanyl-bêta-arnt synthétases
WO2011146410A2 (fr) 2010-05-17 2011-11-24 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic, et d'anticorps associées à des fragments protéiques de leucyl-arnt synthétases
WO2011150279A2 (fr) 2010-05-27 2011-12-01 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps liées à fragments protéiques de glutaminyl-arnt synthétases
WO2011153277A2 (fr) 2010-06-01 2011-12-08 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques, et d'anticorps associés à des fragments de protéine de lysyl-tarn synthétases
JP2012500634A (ja) * 2008-08-29 2012-01-12 シムフォゲン・アクティーゼルスカブ 鳥類から得られた抗体のクローニング方法
WO2012021247A2 (fr) 2010-07-12 2012-02-16 Atyr Pharma, Inc. DÉCOUVERTE INNOVANTE DE COMPOSITIONS THÉRAPEUTIQUES, DE DIAGNOSTIC ET D'ANTICORPS SE RAPPORTANT À DES FRAGMENTS PROTÉIQUES DE GLYCYL-ARNt SYNTHÉTASES
WO2012027611A2 (fr) 2010-08-25 2012-03-01 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et d'anticorps associées à des fragments protéiniques des tyrosyl-arnt synthétases
WO2012163541A1 (fr) 2011-06-01 2012-12-06 Medical Prognosis Institute A/S Procédés et dispositifs pour le pronostic d'une rechute du cancer
WO2013123432A2 (fr) 2012-02-16 2013-08-22 Atyr Pharma, Inc. Histidyl-arnt synthétases pour le traitement de maladies auto-immunes et inflammatoires
WO2014085434A1 (fr) 2012-11-27 2014-06-05 Pontificia Universidad Catolica De Chile Compositions et procédés de diagnostic de tumeurs de la thyroïde
WO2014195032A1 (fr) 2013-06-07 2014-12-11 Medical Prognosis Institute A/S Procédés et dispositifs pour prédire une efficacité de traitement de fulvestrant chez des patients atteints de cancer
WO2016008048A1 (fr) 2014-07-15 2016-01-21 Ontario Institute For Cancer Research Procédés et dispositifs permettant de prédire l'efficacité d'un traitement à l'anthracycline
GB2579856A (en) * 2018-12-18 2020-07-08 Emergex Vaccines Holding Ltd MHC Class I associated peptides for prevention and treatment of hepatitis B virus infection
WO2021205408A1 (fr) * 2020-04-10 2021-10-14 Igy Immune Technologies And Life Sciences Inc. Immunoglobulines igy ciblant le coronavirus, procédés pour les préparer, et procédés les utilisant
CN117821469A (zh) * 2023-11-22 2024-04-05 扬州大学 一种鸡trim45截短体重组蛋白或其多克隆抗体的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0690912B1 (fr) * 1993-04-14 2008-03-05 Commonwealth Scientific And Industrial Research Organisation Vecteur d'adenovirus de recombinaison avien
WO2000029444A1 (fr) * 1998-11-16 2000-05-25 Genway Biotech, Inc. Generation d'anticorps par vaccination polynucleotidique dans le cas d'une espece aviaire

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003054021A3 (fr) * 2001-12-20 2004-02-12 Agricultural Research Council Bibliotheque combinatoire de fragments d'anticorps semi-synthetiques
CN1818651B (zh) * 2006-03-13 2010-07-21 东北农业大学 兔抗鹅IgY+IgY(△Fc)(H+L)辣根过氧化物酶标记抗体
JP2012500634A (ja) * 2008-08-29 2012-01-12 シムフォゲン・アクティーゼルスカブ 鳥類から得られた抗体のクローニング方法
WO2010107825A2 (fr) 2009-03-16 2010-09-23 Pangu Biopharma Limited Compositions et procedes comprenant des variants d'epissage d'histidyl-arnt synthetase presentant des activites biologiques non canoniques
EP3255146A1 (fr) 2009-03-16 2017-12-13 Pangu Biopharma Limited Compositions et procedes comprenant des variants d'epissage d'histidyl-arnt synthetase presentant des activites biologiques non canoniques
WO2010120509A2 (fr) 2009-03-31 2010-10-21 Atyr Pharma, Inc. Compositions et procédés impliquant des aspartyl-arnt synthétases présentant des activités biologiques non canoniques
WO2011139714A2 (fr) 2010-04-26 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps se rapportant à des fragments protéiques de la cystéinyl-arnt synthétase
WO2011139799A2 (fr) 2010-04-27 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps se rapportant à des fragments protéiques d'isoleucyl arnt synthétases
WO2011139853A2 (fr) 2010-04-28 2011-11-10 Atyr Pharma, Inc. Compositions thérapeutiques, diagnostiques et d'anticorps à base de fragments de protéines d'aminoacyl-arnt synthétases
WO2011135459A2 (fr) 2010-04-29 2011-11-03 Medical Prognosis Institute A/S Méthodes et dispositifs permettant de prédire l'efficacité d'un traitement
WO2011139854A2 (fr) 2010-04-29 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à based'anticorps associées à des fragments protéiques d'asparaginyl-arnt-synthétases
WO2011139907A2 (fr) 2010-04-29 2011-11-10 Atyr Pharma, Inc. Découverte innovatrice de compositions thérapeutiques, diagnostiques, et d'anticorps associées aux fragments protéiques des valyle arnt synthésases
WO2011140135A2 (fr) 2010-05-03 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées des fragments protéiques de méthionyl-arnt-synthétases
WO2011139986A2 (fr) 2010-05-03 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps liées à des fragments protéiques d'arginyle-arnt synthétases
WO2011140132A2 (fr) 2010-05-03 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de phénylalanyl-alpha-arnt-synthétases
WO2011140267A2 (fr) 2010-05-04 2011-11-10 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de complexe multi-arnt synthétase p38
WO2011143482A2 (fr) 2010-05-14 2011-11-17 Atyr Pharma, Inc. Découverte de compositions inédites de nature thérapeutique, diagnostique et à base d'anticorps contenant des fragments protéiques de phénylalanyl-bêta-arnt synthétases
WO2011146410A2 (fr) 2010-05-17 2011-11-24 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic, et d'anticorps associées à des fragments protéiques de leucyl-arnt synthétases
WO2011150279A2 (fr) 2010-05-27 2011-12-01 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, de diagnostic et d'anticorps liées à fragments protéiques de glutaminyl-arnt synthétases
WO2011153277A2 (fr) 2010-06-01 2011-12-08 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques, et d'anticorps associés à des fragments de protéine de lysyl-tarn synthétases
WO2012021247A2 (fr) 2010-07-12 2012-02-16 Atyr Pharma, Inc. DÉCOUVERTE INNOVANTE DE COMPOSITIONS THÉRAPEUTIQUES, DE DIAGNOSTIC ET D'ANTICORPS SE RAPPORTANT À DES FRAGMENTS PROTÉIQUES DE GLYCYL-ARNt SYNTHÉTASES
WO2012027611A2 (fr) 2010-08-25 2012-03-01 Atyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et d'anticorps associées à des fragments protéiniques des tyrosyl-arnt synthétases
WO2012163541A1 (fr) 2011-06-01 2012-12-06 Medical Prognosis Institute A/S Procédés et dispositifs pour le pronostic d'une rechute du cancer
WO2013123432A2 (fr) 2012-02-16 2013-08-22 Atyr Pharma, Inc. Histidyl-arnt synthétases pour le traitement de maladies auto-immunes et inflammatoires
EP3311847A1 (fr) 2012-02-16 2018-04-25 Atyr Pharma, Inc. Histidyl-arnt synthétases pour le traitement de maladies auto-immunes et inflammatoires
WO2014085434A1 (fr) 2012-11-27 2014-06-05 Pontificia Universidad Catolica De Chile Compositions et procédés de diagnostic de tumeurs de la thyroïde
WO2014195032A1 (fr) 2013-06-07 2014-12-11 Medical Prognosis Institute A/S Procédés et dispositifs pour prédire une efficacité de traitement de fulvestrant chez des patients atteints de cancer
WO2016008048A1 (fr) 2014-07-15 2016-01-21 Ontario Institute For Cancer Research Procédés et dispositifs permettant de prédire l'efficacité d'un traitement à l'anthracycline
GB2579856A (en) * 2018-12-18 2020-07-08 Emergex Vaccines Holding Ltd MHC Class I associated peptides for prevention and treatment of hepatitis B virus infection
WO2021205408A1 (fr) * 2020-04-10 2021-10-14 Igy Immune Technologies And Life Sciences Inc. Immunoglobulines igy ciblant le coronavirus, procédés pour les préparer, et procédés les utilisant
CN117821469A (zh) * 2023-11-22 2024-04-05 扬州大学 一种鸡trim45截短体重组蛋白或其多克隆抗体的应用

Also Published As

Publication number Publication date
AU2001259631A1 (en) 2001-11-26
WO2001088162A3 (fr) 2002-09-19

Similar Documents

Publication Publication Date Title
WO2001088162A2 (fr) Procedes et vecteurs destines a generer des anticorps dans des especes aviaires et utilisations
US6951742B1 (en) Methods and vectors for generating antibodies in avian species and uses therefor
AU2019279084B2 (en) Diverse antigen binding domains, novel platforms and other enhancements for cellular therapy
US6984488B1 (en) Determination and control of bimolecular interactions
AU2017249424B2 (en) Recombinant arterivirus replicon systems and uses thereof
JP7065907B2 (ja) Ｍ様タンパク質をコードするｍＲＮＡを含む癌ワクチン
KR102281923B1 (ko) 단일 세포들의 초병렬 조합 분석을 위한 시스템들 및 방법들
US6165722A (en) Representations of bimolecular interactions
EP3939606A2 (fr) Composés et procédés améliorés permettant de supprimer des réponses immunitaires à des agents thérapeutiques
JP2010540674A5 (fr)
US12241081B2 (en) B cell receptor modification in B cells
JP2014528001A (ja) アームｐｃｒおよび高処理シーケンシングを用いた抗原特異的適応免疫応答の同定法
AU721832B2 (en) Reading frame independent epitope tagging
WO2002097051A2 (fr) Reseaux de proteines et procedes et systemes pour les produire
Wiehe et al. Mutation-guided vaccine design: a strategy for developing boosting immunogens for HIV broadly neutralizing antibody induction
IL146544A (en) Method of identifying a polynucleotide of a microbe that is expressed in vivo
JP2002510502A (ja) 発現可能な遺伝子配列のライブラリーの産生方法
US20210214722A1 (en) Methods and materials for cloning functional t cell receptors from single t cells
WO1991004327A1 (fr) Modele animal transgenique pour infections virales
Fong et al. Experimental and clinical applications of molecular cell biology in nutrition and metabolism
CN109423500A (zh) 一种Mdr1a/1b双基因敲除的方法及应用
WO2006073627A2 (fr) Acides nucleiques, polypeptides et compositions immunogenes du virus de la vaccine
EP1453965A2 (fr) Vaccin a adn
Four et al. Genomic Organization
HUP0400522A2 (hu) Eljárás emlős epitópokat utánzó anyagok azonosítására

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP