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WO1994009141A1 - Genes codant d'une proteine de surface de p. carini - Google Patents

Genes codant d'une proteine de surface de p. carini Download PDF

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Publication number
WO1994009141A1
WO1994009141A1 PCT/US1993/009635 US9309635W WO9409141A1 WO 1994009141 A1 WO1994009141 A1 WO 1994009141A1 US 9309635 W US9309635 W US 9309635W WO 9409141 A1 WO9409141 A1 WO 9409141A1
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WIPO (PCT)
Prior art keywords
lys
glu
leu
carinii
major surface
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Inventor
Joseph A. Kovacs
C. William Angus
Francoise Powel
Jeffrey C. Edman
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US Department of Health and Human Services
Government of the United States of America
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US Department of Health and Human Services
Government of the United States of America
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Priority to AU54419/94A priority Critical patent/AU5441994A/en
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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
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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

Definitions

  • the present invention relates to the cloning of the major surface antigen of Pneumocystis carinii, a life threatening opportunistic pathogen in HIV-infected patients and the use of that antigen as a vaccine to prevent or control P. carinii infection.
  • P. carinii Although long considered a protozoan, recent molecular biologic studies have shown P. carinii to be a member of the fungi (4-7) .
  • the major surface antigen of P. carinii is a mannose-rich glycoprotein of approximately 110,000 to 120,000 M under reducing and denaturing conditions, with a native M r (molecular weight) of over 300,000 (8-12) .
  • P. carinii isolated from different mammalian species contain similar but antigenically distinct proteins (8,9,13) .
  • the present inventors have recently purified and characterized the major surface protein of both rat (gpll ⁇ ) and human (gp95) P. carinii.
  • the major surface glycoproteins of P. carinii have been found to be the products of a multicopy family of genes.
  • the predicted protein has a MW of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well-conserved hydrophobic region at the carboxy terminus.
  • the presence of multiple related genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is an important mechanism for evading host defenses.
  • the present inventors have isolated and sequenced the
  • It is another object of the invention to provide a method of obtaining a DNA molecule encoding a mammalian ______ carinii major surface glycoprotein which comprises screening a cDNA expression library of P. carinii with an antibody to said major surface glycoprotein to identify positive clones encoding gpll ⁇ and using at least one of said clones or an oligonucleotide probe based on said clones to reveal the presence of multiple genes encoding for said major surface glycoprotein. It is a further object of the invention to provide a mammalian Pneumocystis carinii major surface glycoprotein having the amino acid sequence as shown in Figure lb.
  • It is another object of the invention to provide a vaccine comprising a therapeutically effective amount of a mammaian Pneumocystis carinii major surface glycoprotein or a polypeptide derived therefrom capable of eliciting an immune response to said glycoprotein, and pharmaceutically acceptable parenteral vehicle. It is also an object of the invention to provide a DNA molecule encoding a mammalian Pneumocystis carinii major surface glycoprotein which is a composite (or consensus sequences) of multiple genes which encode said major surface glycoprotein.
  • FIG. 1A-1B Alignment of the deduced amino acid sequences SEQ ID NO: 1 through SEQ ID NO: 7 represent 7 homologous clones encoding the major surface glycoprotein of rat P. carinii. Alignment was performed by the Clustal program of PC-Gene (IntelliGenetics, Inc.) . Cysteine residues are identified in bold. Potential glycosylation sites are underlined. The peptides sequenced directly are shown above the alignment. An * indicates that a residue is conserved among all clones that overlap in that region.
  • M r 116, 000
  • Lyticase treatment solubilizes the major surface glycoprotein, but results in a loss in apparent M r of about 10%.
  • Samples were run on a gradient gel (8% to 16%) prior to transfer to nitrocellulose. Migration of molecular weight markers is indicated on the left.
  • FIG. 3A Southern blot of P. carinii DNA (20 xg/lane) digested with Nde 1 (first lane of each pair) and
  • FIG. 3B Southern blot of P. carinii chromosomes from 5 isolates (lanes 1 to 5) and Saccharomyces cerevisiae chromosomes (lane 6) separated by transverse alternating field electrophoresis (28) and probed with PC5, demonstrating hybridization with multiple P. carinii chromosomes in all isolates. MW, based on S.cerevisiae chromosomes, is indicated on the right.
  • Figure 3C Northern blot of total RNA extracted from 5 isolates (lanes 1 to 5) and Saccharomyces cerevisiae chromosomes (lane 6) separated by transverse alternating field electrophoresis (28) and probed with PC5, demonstrating hybridization with multiple P. carinii chromosomes in all isolates. MW, based on S.cerevisiae chromosomes, is indicated on the right.
  • Figure 3C Northern blot of total RNA extracted from 5 isolates (lanes 1
  • 3T3 cells (10 ⁇ g, lane 1) or 5 P. carinii isolates (5-10 ⁇ g, lanes 2 to 6) probed with PC5. Hybridization to an approximately 4000 bp transcript is seen in P. carinii lanes. Migration of rRNA is indicated on the right.
  • the nucleotide sequence of clone PC3 (SEQ ID NO: 1) encodes for a portion of the coding sequence for the major surface glycoprotein of rat P. carinii.
  • the nucleotide sequence of clone PC5 (SEQ ID NO: 2) encodes for a portion of the coding sequence for the major surface glycoprotein of rat P. carinii.
  • the nucleotide sequence of clone PC14 (SEQ ID NO: 3) encodes for a portion of the coding sequence for the major surface glycoprotein of rat P. carinii.
  • the nucleotide sequence of clone GP3 (SEQ ID NO: 4) encodes for a protein similar to the original gpll6 clones and having a molecular weight of 104,048.
  • nucleotide sequence of clone GP46 (SEQ ID NO: 5) encodes for a portion of the major surface glycoprotein of rat P. carinii.
  • nucleotide sequence of clone GP22 (SEQ ID NO: 6) encodes for a portion of the major surface glycoprotein of rat P. carinii.
  • nucleotide sequence of clone GP14 (SEQ ID NO: 7) encodes for a portion of the major surface glycoprotein of rat P. carinii. DNA (SEQ ID NO: 8) and inferred amino acid sequence
  • SEQ ID NO: 9 illustrate one gene of the major surface glycoprotein of P. carinii.
  • the DNA sequence which was determined from both strands, is a composite of the original GP3 clone (SEQ ID NO: 4) (nucleotides 626 to 3521) and the 5' fragment (1 to 722) that was determined by PCR. Primers used in PCR to identify the 5' end of the sequence are underlined once, and the 5' end of the original clone is underlined twice. The 5' fragment was missing the first nine nucleotides of the 5' primer. The polyadenylation signal is shown in bold. MATERIALS AND METHODS
  • Restriction enzymes were obtained from New England Biolabs (Beverly, MA) .
  • Other enzymes or kits were obtained from Stratagene (La Jolla, CA) , Boehringer Mannheim (Indianapolis, IN) , or InVitrogen (San Diego, CA) .
  • Polymerase chain reaction (PCR 1 ) studies were performed with a DNA thermocycler (Perkin-Elmer/Cetus) using reagents obtained from Perkin-Elmer/Cetus.
  • Radiolabeled chemicals were obtained from New England Nuclear-DuPont (Boston, MA) .
  • Sequenase 2 was obtained from United States Biochemical (Cleveland, OH) .
  • Oligonucleotides- were synthesized on a Cyclone-Plus DNA synthesizer (Milligen Biosearch, Burlington, MA) using reagents obtained from Milligen. Hybond-N+ was obtained from Amersham (Chicago) .
  • Organisms were obtained from immunosuppressed rats and partially purified by Ficoll- Hypaque density gradient centrifugation as described (18) .
  • P. carinii libraries Construction of a P. carinii cDNA library in ⁇ ZAP has been described (4) .
  • a second library was constructed in a similar fashion using oligo- dT-selected mRNA and subcloning into a modified ⁇ ZAP vector (19) , YcDEll, which contained sequences necessary for Saccharomyces cerevisiae replication and expression (Edman, J.C., unpublished observations) . Both libraries were constructed from RNA pooled from three P. carinii preparations.
  • Positive clones were plaque purified, and clones encoding the major surface glycoprotein were identified by the antibody elution technique (21) .
  • approximately 5,000 phage were plated with BB4 cells on NZCYM agar; after 3-4 hours growth at 42°C, plates were overlaid with nitrocellulose that had been soaked in lOmM isopropyl-3-D-thiogalactopyranoside, and incubated overnight.
  • Hybridization was performed overnight at 65°C in 6x SSPE/l%SDS/10x Denhardt's solution (lx SSPE is 0.15 M NaCl, lOmM NaH 2 P0 4 , 1 mM EDTA-Na 2 ,pH 7.4; lx Denhardt's is 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin) . Filters were washed at 65°C in 0.5xSSPE/0.1% SDS. Positive clones were plaque purified, and plasmids (pBluescript plus insert) were rescued according to the manufacturer's instructions (Stratagene) (19) .
  • Inserts were sequenced directly from plasmid using the Sanger dideoxy chain termination method (23) either in the inventors' laboratory using the Sequenase 2.0 kit or commercially (Lofstrand, Gaithersburg, MD) . Polymerase chain reaction (PCR) . The 5' region of GP3 was determined by PCR.
  • oligonucleotide JK58 complementary to positions 306 to 325 of PC3 (SEQ ID NO: 10) (TTAACCGGCCGTGCCATTGC) , which includes the putative initiation codon, was used as a template for reverse transcription (24) , after which the cDNA was tailed with terminal transferase and dGTP, amplified by PCR as described (25) , using primer JK58 and a 1:10 ratio of modified primers ANC SEQ ID NO: 11 (GACTGCATGCGGAAGCTTGGATCCCCCCCCCCCC) and AN (SEQ ID NO: 12) (GACTGCATGCGGAAGCTTGGATCC) , subcloned into pCRlOOO (Invitrogen) , and sequenced.
  • the region 5' to GP3 was then determined by reverse transcription followed by PCR (24) , using a 5' primer corresponding to the previously determined first 20 bases of the mRNA (SEQ ID NO:
  • MSG1 which corresponds to nucleotides 1346 to 1375 of GP3 , is well conserved among the overlapping clones.
  • MSG2 (1573 to 1602 of GP3) and MSG3 (223 to 252 of GP14) are based on regions of PG3 and GP14 that are poorly conserved in other clones.
  • DHPS1 is complementary to 1897 to 1926 of the P_ ⁇ carinii fas gene, which encodes P. carinii dihydropteroate synthase (27) . None of the oligonucleotides contained Eco RI or Nde 1 sites. For pulse-field gels, P.
  • Blots probed with PC5 were prehybridized overnight in 6x SSPE/l%SDS/l0x Denhardt's solution, hybridized overnight at 65°C with [ 32 P] -labeled PC5 (55°C for chromosome blots) , then washed twice for 5 min. at room temperature in 2xSSPE/0.1% SDS followed by two washes for 20 min. at the hybridization temperature in 0.5xSSPE/0.1%SDS or, for chromosomal blots, 0.1xSSPE/0.1%/SDS.
  • Blots probed with oligonucleotides were prehybridized overnight in 2x SSPE/0.5%SDS/5x Denhardt's solution/0.5 ⁇ g/ml sonicated and denatured salmon sperm DNA, hybridized overnight at 60°C with [ 32 P] -labeled oligonucleotide, and washed three times at 60°C for 30 minutes each in 2xSSPE/0.5% SDS.
  • Peptide se ⁇ uencin ⁇ Peptide sequencing was performed (Harvard ⁇ Chem, Cambridge, MA) on peptides of gpll6 following treatment of purified gpll ⁇ (8) with endoproteinase LysC and separation by narrow-bore reverse phase HPLC using previously described techniques (29) .
  • Peptide 1 was selected for sequencing by analyzing the predicted peptide sequences originating between the first two predicted methionines as follows: first, peptide retention prediction suggested such peptides would be retained predominantly in the first quarter of the chromatogram. Second, greater than 70% of the sequences lacked tryptophan or tyrosine and, thus, would be deficient in UV absorbance at 277 nM. On the basis of these two criteria, appropriate fractions were screened by electrospray ionization mass spectrometric analysis for a molecular mass matching a sequence from the desired region (29) .
  • Anti-peptide antibodies The following peptides were synthesized by Peninsula Laboratories (Belmont, CA) : GP3 46 . 460 (SEQ. I.D. NO. :9, amino acids 446-460) (Glu-Leu-Lys-Gly- Lys-Leu-Gly-His-Val-Arg-Phe-Tyr-Ser-Asp-Pro) , which corresponds to amino acid residues 446-460 of GP3 and 453 to 467 of PC3; and PC5 365 _ 379 (SEQ. I.D. NO.
  • M r Molecular weight Determinations.
  • the M r for purified major surface glycoprotein was determined for the native protein by a sizing column used on an HPLC, and for the reduced and denaturated protein by SDS-polyacrylamide gel electrophoresis.
  • the M r was determined by the computer program MacVector, based on the amino acid sequences of the predicted proteins.
  • the composite was generated by alignment of the sequence of the original GP3 clone and the sequence of the PCR-generated fragment corresponding to the 5' end of the gene. Nucleotides 626 to 722 of the PCR fragment were found to be identical to nucleotides 1 to 96 of the original GP3 clone. This allowed appending nucleotides 1 to 625 of the PCR fragment to the 5' end of GP3, to generate the composite full-length clone. Consensus sequence synthesized gene. A consensus sequence can be generated by computer alignment of the proteins encoded by each gene from the multiple clones .
  • a clone containing a synthetic construction and representing the consensus sequences, or regions that contain some of the consensus sequences, could then be derived by molecular biologic techniques, for example by replacing regions in one of the clones with consensus sequences, or by using site-directed mutagenesis (39) .
  • the clone with the largest insert (GP3) , 2869 bases plus a poly-A tail, has an open reading frame encoding for a protein of 104,048 MW. Based on the sequences in the composite protein, GP3 also appeared to be incomplete at the 5' end; PCR was utilized to determine the full sequence of this gene. The 5' end of the message was identified by anchored PCR (24) using primer JK58, which spanned the putative start codon of the composite protein. The intervening region was determined by reverse transcription followed by PCR, using primers spanning the 5' end to base 722 in GP3. A single clone was identified that had an identical sequence to the first 76 bases of GP3. The complete, composite cDNA contained an open reading frame encoding a protein of 122,997 MW.
  • Peptides based on regions in GP3 and PC5 were used to generate antibodies in rabbits. By immunoblot, these anti- peptide antibodies were shown to react with intact as well as lyticase-solubilized gpll6 (See Figure 2) .
  • Antigenic variability of surface proteins is an important mechanism for evading host defenses in a number of organisms (e.g., trypanosome and borrelia species) (31,32) .
  • the present inventors Upon the identification of multiple clones encoding for a family of closely related surface proteins, the present inventors theorized that antigenic variability of surface proteins may be important to P. carinii.
  • Heterogeneity of genes for gpll ⁇ may represent the occurrence of multiple alleles for a single-copy gene, multiple genes encoding for a family of related proteins, splicing, or a combination of these factors.
  • the genes encoding gpll ⁇ are rich in adenosine and thymidine residues (63% in GP3) , and is 69% adenosine or thymidine in the third codon position of GP3, similar to the other coding sequences of P. carinii that have been identified to date (4,5) .
  • Nucleotide variability among the clones is not located primarily in the wobble position of the codon: among the 437 nucleotides that differ in PC14 and GP3, for example, 36% are in the first, 30% in the second, and 34% in the third codon position.
  • GP3 has a consensus polyadenylation signal (AATAAA) at nucleotides 3470-3475.
  • cysteine residues do not occur at random, but are most often separated from another cysteine by six (20 occurrences) or seven (six occurrences) amino acids. There is no predictable pattern to the intervening amino acids.
  • a region rich in threonine and serine residues near the carboxy terminus (953-1052 of GP3) are poorly conserved region rich in proline and glycine residues between residues 817 and 870 of GP3, and a region rich in threonine and serine residues near the carboxy terminus (953-1052 of GP3) .
  • gpll6 has N-linked carbohydrate residues that account for approximately 10% of its apparent molecular weight.
  • GP3 contains five potential glycosylation sites (Asn-X-Ser/Thr) ( Figure la) . Two of these sites (573 and 809) are conserved in the overlapping regions of the other clones. It is unknown whether O- linked glycosylation sites also exist in gpll6.
  • the threonine/serine-rich region may be a candidate for such glycosylation, as has been suggested for a serine-rich region in yeast gpll5 and a threonine rich region in the promastigote surface antigen-2 of Leishmania major (33,34) .
  • the DNA sequences for the P. carinii major surface glycoprotein can be modified to provide sequences that are mutants, deletions, or substitutions thereof which encode a protein having at least 90% homology with the naturally occurring major surface glycoprotein and possessing substantially the same properties as the P. carinii major surface glycoprotein.
  • the major surface glycoproteins of P. carinii preferably comprises one of a homologous variant of said major surface glycoproteins of P. carinii having less than 8 conservative amino acid changes, preferably less than 5 conservative amino acid changes.
  • conservative amino aid changes are substitutions of one amino acid by another amino acid wherein the charge and polarity of the two amino acids are not fundamentally different .
  • Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) neutral polar amino acids, (3) neutral non-polar amino acids and (4) basic amino acids. Conservative amino acid changes can be made by substituting one amino acid within a group by another amino acid within the same group. Representative amino acids within these groups include, but are not limited to, (1) acidic amino acids such as aspartic acid and glutamic acid, (2) neutral polar amino acids such as valine, isoleucine and leucine, (3) neutral nonpolar amino acids such as asparganine and glutamine and (4) basic amino acids such as lysine, arginine and histidine. In addition to the above mentioned substitutions, the major surface glycoproteins of P.
  • carinii of the present invention may comprise the above mentioned specific amino acid sequences and additional sequences at the N-terminal end, C-terminal end or in the middle thereof.
  • the "gene" or nucleotide sequence may have similar substitutions which allow it to code for the corresponding major surface glycoproteins of P. carinii.
  • Individual base pair changes or deletions or insertion of the DNA encoding for the major glycoproteins of P. carinii can be made by the methods of site-directed mutagenesis which are well known in the art. See Sambrook et al (39) .
  • carinii is ligated into a replicable (reproducible) vector, the vector is used to transform host cells, and the affector is recovered from the culture.
  • the host cells for the above-described vectors include gram-negative bacteria such as E. coli , gram-positive bacteria, yeast and mammalian cells. Suitable replicable vectors will be selected depending upon the particular host cell chosen.
  • P. carinii has been a major pathogen in human immunodeficiency virus of infected patients since the beginning of the AIDS epidemic, inability to culture the organism has made studies of immunopathogenesis very difficult.
  • Experiments investigating host-organism interactions have recently focused on the major surface glycoprotein. Although the function of this protein is unknown, it is an abundant surface-exposed glycoprotein that has the potential to interact with multiple host cell- associated or secreted proteins. As a surface protein, it is likely a primary target of the immune response.
  • the present inventors have shown in the current experiments that multiple genes encode a family of related major surface glycoproteins, and that, based on chromosomal blots, multiple copies of these genes are present in the P. carinii genome.
  • antigenic variability may play a role in immune evasion.
  • antigenic variability is well-known in protozoal and bacterial pathogens (31,32)
  • the variability of the major surface glycoprotein is the first description of this phenomenon in the fungi .
  • the corresponding human antigen can be prepared as follows:
  • P. carinii organisms could be obtained from human HIV- infected patients and partially purified by Ficoll-Hypaque density gradient centrifugation as described (18) .
  • Human P. carinii libraries could be constructed in the same manner as the P. carinii cDNA library in ⁇ ZAP that has been described (4) .
  • a second library can be constructed in a similar fashion using oligo-dT-selected mRNA and subcloning into a modified ⁇ ZAP vector (19) , YcDEll, which contained sequences necessary for Saccharomyces cerevisiae replication and expression.
  • YcDEll modified ⁇ ZAP vector (19) , YcDEll
  • the library could be screened with the already identified rat P. carinii surface antigen genes. This could identify the genes since antibody studies have demonstrated that although the rat and human P. carinii proteins are antigenically different, there is also cross- reactivity, and thus, there is likely to be conservation at the DNA level as well. Once one human P. carinii major surface glycoprotein gene is identified, that gene may be used to identify other members of the gene family.
  • the library may be screened using a conserved oligonucleotide whose sequence is based on the available rat P. carinii major surface glycoprotein genes. Since conserved regions are presumably functionally important, and since the rat and human P. carinii major surface glycoprotein are homologous, they would have conserved the same regions that were conserved among rat P. carinii genes . Low stringency conditions may be used to obtain hybridization even if the conservation is not absolute. 3.
  • conserved oligonucleotides may be used based on sequences of the rat P. carinii major surface glycoprotein genes as primers for the polymerase chain reaction to be performed using human P. carinii DNA extracts as template. Conditions may be adjusted to low stringency if needed. Once a human P. carinii-specific piece of DNA is amplified, that DNA fragment may then be used to screen the library to identify larger fragments or the entire gene.
  • Amino-acid sequence information from the purified human P. carinii major surface glycoprotein may be obtained by direct sequencing of proteolytic-enzyme generated fragments, in a manner similar to that done with the rat P. carinii major surface glycoprotein. This information may then be used to generate oligonucleotides that may be used either directly to screen the library, or as primers for PCR to amplify a fragment of the human P. carinii major surface glycoprotein gene, which may then be used for further screening.
  • P. carinii has been one of the most devastating complications of the immunosuppression associated with human immunodeficiency virus infection.
  • the use of chemoprophylaxis has lead to a marked decline in the incidence of P. carinii pneumonia, but the agents used for prophylaxis are associated with significant adverse reactions or a high failure rate (37) .
  • the recent demonstration that novel, potentially protective, immune responses to HIV can be induced by immunization of HIV- infected patients with rgpl60 (38) suggests that immunoprophylaxis may also be an effective alternative for controlling HIV-related opportunistic infections.
  • the major surface glycoprotein of P. carinii can be used as a vaccine and as a diagnostic reagent. Additionally, the detailed study of this protein and its expression should lead to an understanding of its functional role in the pathogenesis of P. carinii pneumonia, and may lead to novel strategies designed to prevent or control P. carinii infection and its devastating consequences.
  • the P. carinii major surface glycoprotein antigen of this invention can be administered to mammals; e.g., human, in a variety of ways.
  • exemplary methods include parenteral (subcutaneous) administration given with a nontoxic adjuvant, such as an alum precipitate or peroral administration given after reduction or ablation of gastric activity; or in a pharmaceutical form that protects the antigen against inactivation by gastric juice (e.g., a protective capsule or microsphere) .
  • the dose and dosage regimen will depend mainly upon whether the antigen is being administered for therapeutic or prophylactic purposes, the patient, and the patient's history.
  • the total pharmaceutically effective amount of antigen administered per dose will typically be in the range of about 5 ⁇ g to 1280 ⁇ g per patient.
  • the antigen will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate, may also be used.
  • Liposomes may be used as vehicles.
  • the vehicle may contain minor amounts of additives, such as substances which enhance isotonicity and chemical stability; e.g., buffers and preservatives.
  • the recombinant major surface glycoprotein of this invention can provide a reagent to be used in a variety of diagnostic assays to detect antibodies to P. carinii as well as being useful in developing additional reagents that can detect antigens in clinical specimens.
  • the recombinant protein can be used directly in assays to detect anti-P. carinii antibodies.
  • assays would include, for example, ELISA (enzyme-linked immunosorbent assays) , western blot (immunoblot) and immunoprecipitation assays.
  • antibodies either polyclonal or monoclonal antibodies, can be generated to the recombinant proteins. These antibodies can then be used in antigen- capture assays using, for example, an ELISA format, and in immunofluorescent assays.
  • sequences of the genes can also be used to make primers for use in polymerase chain reaction studies for the diagnosis of P. carinii infection as well as to make oligonucleotide probes that can be used directly in diagnostic assays for detecting the DNA of P. carinii.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carinii
  • ATGTTTTTAC ATGTTTTTAC TGATGGTTAT CCTCGTGGAG GTCATTTGAT CGAGGATGAG TTGTCCGAAG 300
  • AATGTATACT TTTAGAAGAC ACGGGTTATA GTGAAGATAT TAAGAAGAAC TGTGTCAAGT 720
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carinii
  • AAACCTTTAG AAACAGAATT GAATGAAA AGCTCAGAAA AGTGTCATGA AAGACTTGAG 4
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carnii
  • GAAGTAGAAG AACTCTGCAC AACATGGGGT AAATATTGTC ACCAACTTAT GGAGAATTGT 84
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carnii
  • AGGATTGTCT ACGGAATATG ATGATCAAGA ATCAGATCCT TTATCGTGGG GACAGCTGCC 132
  • AAAGATGAAG AATGAGTACG ATGATCTCAA AAAGGCGGCA GAAAAATCTA CGGAGGCAGC 252
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carnii
  • AAAGCAAATG AAACAGCACT CGAAGAACTC TGCACAACAT GGGGCCGACA TTGTCACCAA 66
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carnii
  • AAGCCTCGAC AAACCGTAAC GCCAAATGCG CAGAATGGCA GTGCTTCTGG ACCAGTACCA 126
  • CCAAACACAA CAGATGAAAC GACGAAGAAT CCAAGCCTTG GACTCGTTAA AAGAGCATAT 144
  • AAAAAAAAAA 211 INFORMATION FOR SEQ ID NO:7 :
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carnii
  • GAAGCATGTC AAAATGCACG AGCAGCGTGC TATAAAGTGG GAAAAGATAG GATGTTGAGT 12
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pneumocystis carnii
  • AAG CGT GAA AAG GTG GCA GAG GAG CTT CTT CTG AGG GCG CTC GGA GGG 60 Lys Arg Glu Lys Val Ala Glu Glu Leu Leu Leu Arg Ala Leu Gly Gly 140 145 150
  • GGT TTT AAA GAG GAT TGT CCA GAT ACT AAA CAA GCT TGC GAA AAT ATA 295 Gly Phe Lys Glu Asp Cys Pro Asp Thr Lys Gin Ala Cys Glu Asn He 925 930 935
  • GAG AAA ATA ACA GAA ACA AAG ACG GAA ACG AAG ACG GAA ACA AAG ACG 305 Glu Lys He Thr Glu Thr Lys Thr Glu Thr Lys Thr Glu Thr Lys Thr 955 960 965
  • AAAATGTGCA TATATCCATT GTTTATATAT AATAAAAATG TTAAAGAATG AAATGAAAAA 349
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

On a cloné et séquencé sept gènes uniques codant la principale glycoprotéine de surface du P. carinii murin associée au principal antigène de surface de P. carinii, qui est un germe pathogène opportuniste mortels, chez des patients infectés par le VIH. On peut préparer des gènes codant la principale glycoprotéine de surface du P. carinii humain et des vaccins contenant l'antigène de P. carinii humain afin de prévenir ou de combattre l'infection par P. carinii.
PCT/US1993/009635 1992-10-09 1993-10-08 Genes codant d'une proteine de surface de p. carini Ceased WO1994009141A1 (fr)

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AU54419/94A AU5441994A (en) 1992-10-09 1993-10-08 Genes that encode a surface protein of p. carinni

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US95868392A 1992-10-09 1992-10-09
US07/958,683 1992-10-09

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Publication number Priority date Publication date Assignee Title
US6664053B1 (en) 1998-08-17 2003-12-16 The United States Of America As Represented By The Department Of Health And Human Services Identification of a region of the major surface glycoprotein (MSG) gene of human Pneumocystis carinii

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Publication number Priority date Publication date Assignee Title
WO1993007274A1 (fr) * 1991-09-30 1993-04-15 The General Hospital Corporation Clonage moleculaire d'antigenes de surface de pneumocystis carinii et leur utilisation pour la detection de pneumocystis carinii

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WO1993007274A1 (fr) * 1991-09-30 1993-04-15 The General Hospital Corporation Clonage moleculaire d'antigenes de surface de pneumocystis carinii et leur utilisation pour la detection de pneumocystis carinii

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6664053B1 (en) 1998-08-17 2003-12-16 The United States Of America As Represented By The Department Of Health And Human Services Identification of a region of the major surface glycoprotein (MSG) gene of human Pneumocystis carinii

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