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WO2001088151A1 - Facteur 30 de regulation de transcription de microglobuline, polypeptide humain, et polynucleotide le codant - Google Patents

Facteur 30 de regulation de transcription de microglobuline, polypeptide humain, et polynucleotide le codant Download PDF

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Publication number
WO2001088151A1
WO2001088151A1 PCT/CN2001/000709 CN0100709W WO0188151A1 WO 2001088151 A1 WO2001088151 A1 WO 2001088151A1 CN 0100709 W CN0100709 W CN 0100709W WO 0188151 A1 WO0188151 A1 WO 0188151A1
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Prior art keywords
polypeptide
polynucleotide
regulatory factor
human microglobulin
sequence
Prior art date
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PCT/CN2001/000709
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU70450/01A priority Critical patent/AU7045001A/en
Publication of WO2001088151A1 publication Critical patent/WO2001088151A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human microglobulin transcription regulation factor 30, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • this gene may also be a reverse transcript, because its genetic structure has two short forward repeats, a polyadenylation tail signal and a polyadenylation tail (Mamm Genome, 9 (2 ): 103-106).
  • Class I major histocompatibility antigen and human P 2 microglobulin are non-covalently linked on the cell membrane, and play an important role in T-cell antigen recognition (Advance in Immunology, 27: 51-177), and allogeneic Or immune rejection during xenotransplantation is also closely related.
  • the human microglobulin transcriptional regulatory factor 30 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more participation in the field. These processes of the human microglobulin transcription factor 30 protein, in particular, identify the amino acid sequence of this protein.
  • New human microglobulin transcription factor 30 egg Isolation of the white-coding gene also provides a basis for research to determine the role of the protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so isolating its coding for DM is very important.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human microglobulin transcriptional regulatory factor 30. '
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human microglobulin transcriptional regulatory factor 30.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide-human microglobulin transcription regulation factor 30 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities in human microglobulin transcriptional regulator 30. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 99-908 in SEQ ID NO: 1; and (b) a sequence having 1-1604 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing a polynucleotide of the invention;
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell; a method for preparing a polypeptide of the present invention comprising culturing the host cell and recovering an expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human microglobulin transcriptional regulatory factor 30 protein, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or disease susceptibility associated with abnormal expression of human microglobulin transcriptional regulatory factor 30 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or Detection of the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human microglobulin transcriptional regulatory factor 30.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human microglobulin transcription regulatory factor 30 and human microglobulin transcription regulatory factor 34 according to the present invention.
  • the upper graph is a graph of the expression profile of human microglobulin transcriptional regulatory factor 30, and the lower graph is the graph of the expression profile of human microglobulin transcriptional regulatory factor 34.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates non-starved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 Indicates prostate
  • 21 indicates fetal heart
  • 22 indicates heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human microglobulin transcription regulatory factor 30.
  • 30kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human microglobulin transcriptional regulatory factor 30, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human microglobulin transcriptional regulatory factor 30.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human microglobulin transcription regulatory factor 30 when combined with human microglobulin transcription regulatory factor 30.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human microglobulin transcriptional regulatory factor 30.
  • Regular refers to a change in the function of human microglobulin transcription regulatory factor 30, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human microglobulin transcription regulatory factor 30 Change of nature.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Quality. Those skilled in the art can purify human microglobulin transcriptional regulatory factors using standard protein purification techniques
  • the substantially pure human microglobulin transcriptional regulatory factor 30 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human microglobulin transcriptional regulatory factor 30 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction. '
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method divides each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the assay may be Jotun Hein percent identity between nucleic acid sequences Clus ter or a method well known in the art (Hein J., (1990) Methods in enzyraology 183: 625-645) 0
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? 7. It can specifically bind to the epitope of human microglobulin transcriptional regulatory factor 30.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human microglobulin transcription regulatory factor 30 means that human microglobulin transcription regulatory factor 30 is substantially free of other proteins, lipids, sugars, or other substances that are naturally associated with it.
  • Those skilled in the art can purify human microglobulin transcriptional regulatory factor 30 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human microglobulin transcription factor 30 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide ⁇ ⁇ microglobulin transcription regulation factor 30, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • the invention also includes fragments, derivatives, and analogs of human microglobulin transcription regulatory factor 30.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human microglobulin transcriptional regulatory factor 30 of the present invention.
  • Polypeptides of the invention may be: (I) a kind in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues, preferably conservative amino acid residues, And the substituted amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ I) a method in which a mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a method in which an additional amino acid sequence is fused into a mature polypeptide Polypeptide sequence (such as the leader or secretory sequence or the sequence used to purify the polypeptide or protein sequence).
  • fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1604 bases, and its open reading frames 99-908 encode 269 amino acids.
  • this polypeptide has a similar expression profile with human microglobulin transcriptional regulatory factor 34, and it can be deduced that the human microglobulin transcriptional regulatory factor 30 has a similar function as human microglobulin transcriptional regulatory factor 34.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not Change the function of the polypeptide it encodes.
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • the "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides. Nucleic acid fragments and above. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human microglobulin transcriptional regulatory factor 30.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human microglobulin transcription regulatory factor 30 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or CDM libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDM of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDM library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Labora tory Manua, Cold Spruing Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be screened from these CDM libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of transcripts of human microglobulin transcriptional regulatory factor 30; (4) through immunological techniques or determination of health Physical activity to detect protein products expressed by genes. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and is at least 10 nucleotides in length, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human microglobulin transcriptional regulatory factor 30 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DM / RM using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as a dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 546 3-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human microglobulin transcription regulatory factor 30 coding sequence, and produced by recombinant technology. Said method of polypeptide.
  • a polynucleotide sequence encoding a human microglobulin transcription regulatory factor 30 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • MSXND expression vectors expressed in mammalian cells Lee and Nathans, J Bio Chera. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells In short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Expression vector with 30 DM sequences and appropriate transcriptional / translational regulatory elements include in vitro recombination DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989).
  • the DM sequence can be operably linked to an appropriate promoter in an expression vector to guide fflRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human microglobulin transcription regulation factor 30 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells, etc.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM may be in exponential growth phase were harvested after the treatment with (Method 12, using the procedure well known in the art. Alternatively, it is a MgCl 2. If If required, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, Or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human microglobulin transcription regulatory factor 30 (Science, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Class I major histocompatibility antigen genes and ⁇ 2 microglobulin genes have both common gene regulatory units that are synergistic in expression, and these two genes differ in expression regulation. Class I major histocompatibility antigens are important markers of cell-to-cell recognition and are important for graft rejection and resistance to disease.
  • the ⁇ 2 microglobulin transcriptional regulator HCNGP has been found to be a DM binding protein present in the downstream sequence of the rat microglobulin gene. It exists in the nucleus and plays an important role in regulating the expression of microglobulin genes. At the same time, this gene may also be a reverse transcript.
  • Class I major histocompatibility antigens and human P 2 microglobulin are non-covalently linked on the cell membrane.
  • ⁇ 2 microglobulin transcriptional regulatory factors are involved in the immune response due to the transcriptional regulation of ⁇ 2 microglobulin.
  • T-cell antigen recognition plays an important role in enabling T killer cells to exert effective killing effects.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human ⁇ 2 microglobulin transcription regulator, Both have similar biological functions. It is involved in immune response and immune monitoring in the body, and is extremely necessary for allogeneic cells, for transplant organ cells, and for recognition of viral antigens. Its abnormal expression is usually closely related to the occurrence of the above-mentioned dysfunction of the tissue system, and causes related diseases.
  • the abnormal expression of the human microglobulin transcriptional regulatory factor 30 of the present invention will produce various diseases, especially immune graft rejection, viral infectious inflammation, other immune diseases, inflammation, various tumors, these diseases including but not limited to:
  • Immune graft rejection Kidney transplant rejection, Heart transplant rejection, Transfusion reaction, Viral infection: Acquired immunodeficiency, Japanese encephalitis, Hepatitis A, Acute measles, Varicella, Mumps, Spinal cord Poliovirus, epidemic hemorrhagic fever
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B lymphocyte immunodeficiency disease, Primary T lymphocyte immunodeficiency disease
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Tumors of various tissues thymic tumor, esophageal cancer, breast cancer, leukemia, lymphoma, gastric cancer, liver cancer, lung cancer, thyroid tumor, uterine fibroids, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, bladder cancer, endometrial cancer, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma
  • Abnormal expression of the human microglobulin transcriptional regulatory factor 30 of the present invention will also produce certain hereditary, hematological diseases, and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used for the treatment of diseases, for example, it can treat various diseases, especially immune graft rejection, viral infectious inflammation, other immune diseases, inflammation , Various tumors, some hereditary, hematological diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human microglobulin transcriptional regulatory factors 30.
  • Agonists enhance human microglobulin transcription regulatory factors 30 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human microglobulin transcription regulation factor 30 and labeled human microglobulin transcription regulation factor 30 can be cultured together in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human microglobulin transcription regulator 30 include screened antibodies, compounds, and receptors Deletions and analogs, etc. Antagonists of human microglobulin transcription regulatory factor 30 can bind to human microglobulin transcription regulatory factor 30 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot function biological functions.
  • human microglobulin transcription regulatory factor 30 can be added to the bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between human microglobulin transcription regulatory factor 30 and its receptor. Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human microglobulin transcription regulator 30 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 30 molecules of human microglobulin transcriptional regulatory factors should be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides antibodies against human microglobulin transcriptional regulatory factor 30 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human microglobulin transcription regulatory factor 30 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc.
  • Factor 30 monoclonal antibodies include, but are not limited to, hybridoma technology (Kohl er and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • An inlay antibody combining a human constant region and a non-human variable region can be produced using existing technologies (Morrison et al, PMS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human microglobulin transcription regulatory factor 30.
  • Antibodies against human microglobulin transcription regulatory factor 30 can be used in immunohistochemical techniques to detect human microglobulin transcription regulatory factor 30 in biopsy specimens.
  • Monoclonal antibodies that bind to human microglobulin transcriptional regulatory factor 3'0 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human microglobulin transcription regulatory factor 30 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill Human microglobulin transcription factor 30 positive cells
  • the antibodies of the present invention can be used to treat or prevent diseases related to human microglobulin transcriptional regulatory factor 30.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human microglobulin transcriptional regulatory factor 30.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human microglobulin transcriptional regulatory factor 30.
  • diagnostic tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human microglobulin transcription regulatory factor 30 detected in the test can be used to explain the importance of human microglobulin transcription regulatory factor 30 in various diseases and to diagnose the role of human microglobulin transcription regulatory factor 30. disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human microglobulin transcription regulatory factor 30 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / non-active expression of human microglobulin transcription regulatory factor 30.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human microglobulin transcription regulatory factor 30 to inhibit endogenous human microglobulin transcription regulatory factor 30 activity.
  • a mutated human microglobulin transcriptional regulatory factor 30 may be a shortened human microglobulin transcriptional regulatory factor 30 that lacks a signaling functional domain. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human microglobulin transcription regulatory factor 30.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human microglobulin transcription regulatory factor 30 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human microglobulin transcriptional regulatory factor 30 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human microglobulin transcription regulatory factor 30 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RM and DNA
  • ribozymes that inhibit human microglobulin transcription regulatory factor 30 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like R molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RM and DM and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid phase phosphorus The technology of synthesizing oligonucleotides by acid amide chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DM sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human microglobulin transcription regulator 30 can be used for the diagnosis of diseases related to human microglobulin transcription regulator 30.
  • the polynucleotide encoding human microglobulin transcription regulatory factor 30 can be used to detect the expression of human microglobulin transcription regulatory factor 30 or the abnormal expression of human microglobulin transcription regulatory factor 30 in a disease state.
  • a DNA sequence encoding human microglobulin transcription regulatory factor 30 can be used to hybridize biopsy specimens to determine the expression status of human microglobulin transcription regulatory factor 30.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human microglobulin transcription regulatory factor 30 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification. Human microglobulin transcription regulatory factor 30 transcription products can also be detected.
  • Human microglobulin transcription regulatory factor 30 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human microglobulin transcription regulatory factor 30 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize MA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome can be utilized Or a large number of genomic clones to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific CDM library.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technologies, the cD that is accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human microglobulin transcription regulator 30 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human microglobulin transcription regulatory factor 30 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription.
  • a Smart cDNA cloning kit purchased from Clontech was used to orient the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a, and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with a public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0276g07 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0276g07 clone contained a full-length cDNA of 1604bp (as shown in Seq ID NO: 1), and a 809bp open reading frame (0RF) from 99bp to 908bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0276 g 07 and the encoded protein was named human microglobulin transcriptional regulatory factor 30.
  • Example 2 Cloning of a gene encoding human microglobulin transcription regulatory factor 30 using RT-PCR method. Fetal brain cells were used as a template, and ol igo-dT was used as a primer for reverse transcription reaction to synthesize cDM. A kit for Q agene was used. After purification, PCR amplification was performed with the following primers:
  • Primer 1 5
  • SEQ ID NO: 3 5
  • Pr iraer2 5'- AAATAATTTTATTTACAGAAAATT -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • a reaction volume of 50 ⁇ contains 5 Ommo 1 / L KC1, 10 mmol / L Tri s-HCl pH 8. 5, 1. 5 and ol / L MgCl 2 , 20 ( ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (product of Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C. 2min. Set ⁇ -act in as a positive control and template blank as a negative control at the same time during RT-PCR.
  • Amplification products were purified with QIAGEN kit and TA The cloning kit was ligated to a pCR vector (Invitrogen). The DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-1604bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human microglobulin transcriptional regulatory factor 30 gene expression Total RNA was extracted in one step [Anal. Biochera 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) Centrifuge after mixing. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA containing 20raM 3- (N- morpholino) propanesulfonic acid (pH7 0.) - subjected to electrophoresis on a 1.2% agarose gel 5mM sodium acetate -IraM EDTA-2 2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • a 32 P dATP was used to prepare 32 P-labeled DM probes by random primer method.
  • the DM probe used is the PCR amplified human microglobulin transcription regulatory factor 30 coding region sequence (99bp to 908bp) shown in FIG. 1.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and an RNA-transferred nitrocellulose membrane were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 50% formamide-25mM KH 2 P0 4 (pH7.4)-5 x SSC-5 x Denhardt, s solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, filter membrane Wash in 1 x SSC-0. 1% SDS at 55 ° C. for 30 min. Then, analyze and quantify with Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human microglobulin transcription regulatory factor 30 according to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:
  • Pr imer3 5,-CATCCATGGATGAATCAGGAGGATCTAGATCCG -3 '(Seq ID No: 5)
  • Pr imer4 5'-CATGGATCCCCCTGATGGAAAGCTTCTAATCT -3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively. Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • PCR reaction was performed using pBS-0276g07 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS-0276gO7 containing 10pg, primers Pr imer-3 and Pr imer-4 were l Opmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1.
  • Cycle parameters 94. C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E.
  • coli DH5c by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ⁇ g / ml), positive clones were selected by colony PCR method and sequenced. Select positive clones (PET-0276g07) with correct sequence Granules were transformed into E. coli BL21 (DE3) plySs (product of Novagen). Containing kanamycin (final concentration of 3 ( ⁇ g / ml) of LB liquid medium, host strain BL21 (P ET-0276g07) cultured at 37 ° C to logarithmic phase, IPTG was added to a final concentration of lmmol / L, continue to culture for 5 hours.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human microglobulin transcription regulatory factor 30-specific peptides:
  • NH2-Met-Asn-Gln-Glu-Asp-Leu-Asp-Pro-Asp-Ser-Thr-Thr-Asp-Val-Gly-C00H (SEQ ID NO: 7).
  • the polypeptide was coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4rag jk cyanin polypeptide complex and complete Freund's adjuvant, and 15 days later, hemocyanin polypeptide complex and incomplete Freund's adjuvant were used to boost immunity once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • Filter membrane hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods, etc., all of which fix the polynucleotide sample to be tested on the filter The membranes were hybridized using essentially the same procedure.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements For homology comparison of the regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (bond) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, in order to follow the experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32P_dATP) is prepared.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRi si, JL, Lyer, V.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs as target DNA, including the present invention Polynucleotide. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification.
  • the Cartesian 7500 spotter purchased from Cartesian Company, USA
  • the spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DM on the glass slide to prepare chips.
  • the specific method steps have been reported in the literature.
  • the sample post-processing steps in this embodiment are:
  • Total mRM was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified with Ol igotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5-Amino-propar gy 1-2 '-deoxyur dine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP ( 5-Amino-propargyl-2'-deoxyuridine 5--triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) in the body, and probes were prepared after purification.
  • Cy3dUTP 5-Amino-propar gy 1-2 '-deoxyur dine 5'-triphate coupled to Cy3 f lu
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridizat ion Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen , Fetal brain, fetal lung, and fetal heart.

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Abstract

L'invention concerne un polypeptide humain, un facteur 30 de régulation de transcription de microglobuline, le polynucléotide qui le code et le procédé de production de ce polypeptide à l'aide de technologie de recombinaison de l'ADN. Elle concerne aussi des méthodes de traitement de nombreuses maladies, telles que tumeur maligne, hémopathie, infection VIH, maladie immunologique, inflammations variées, et d'autres, par utilisation du polypeptide. Elle concerne encore l'antagoniste du polypeptide et son utilisation thérapeutique. Elle concerne enfin l'utilisation du polynucléotide codant un facteur 30 de régulation de transcription de microglobuline humain.
PCT/CN2001/000709 2000-05-09 2001-05-08 Facteur 30 de regulation de transcription de microglobuline, polypeptide humain, et polynucleotide le codant Ceased WO2001088151A1 (fr)

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CN 00115610 CN1322735A (zh) 2000-05-09 2000-05-09 一种新的多肽——人微球蛋白转录调控因子30和编码这种多肽的多核苷酸

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6011146A (en) * 1991-11-15 2000-01-04 Institut Pasteur Altered major histocompatibility complex (MHC) determinant and methods of using the determinant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6011146A (en) * 1991-11-15 2000-01-04 Institut Pasteur Altered major histocompatibility complex (MHC) determinant and methods of using the determinant

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