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WO1994011505A1 - Polypeptides specifiques de pars planitis - Google Patents

Polypeptides specifiques de pars planitis Download PDF

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Publication number
WO1994011505A1
WO1994011505A1 PCT/US1993/010825 US9310825W WO9411505A1 WO 1994011505 A1 WO1994011505 A1 WO 1994011505A1 US 9310825 W US9310825 W US 9310825W WO 9411505 A1 WO9411505 A1 WO 9411505A1
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Prior art keywords
antibody
sequence
cells
expression vector
cell
Prior art date
Application number
PCT/US1993/010825
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English (en)
Inventor
Nalini S. Bora
Henry J. Kaplan
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Washington University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by Washington University filed Critical Washington University
Priority to AU58963/94A priority Critical patent/AU5896394A/en
Publication of WO1994011505A1 publication Critical patent/WO1994011505A1/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This invention relates to the field of ophthalmology. More particularly, it relates to novel polypeptides and methods for the diagnosis of pars planitis , an inflammatory condition of the eye. In a further aspect, the invention concerns the production of pars planitis specific polypeptides by recombinant DNA techniques .
  • a leading cause of blindness in the United States is intraocular inflammation specifically uveitis (Bloch-Michel, Am. J. Qphthalmol. 103 (1987), pg. 234).
  • the most difficult question facing the ophthalmologist when encountering a patient with uveitis is the cause of the ocular inflammation. Without a definite etiologic diagnosis the patient can only be treated and not cured.
  • Pars planitis is a relatively common, clinically well-defined form of uveitis whose etiology is unknown.
  • the disease takes its name from the fact that inflammation affects an area of the eye, the pars plana, that is intermediate between the anterior and posterior uvea .
  • it is a type of intermediate uveitis.
  • It is a chronic inflammatory disease whose onset is generally insidious (Kraus-Macki , et al . , (eds. ) , Uveitis; Pathophysiolo ⁇ v and Therapy, Georg Thieme Verlag, New York, pgs . 98-100).
  • Pars planitis has traditionally been diagnosed by clinical examination with the benefit of a comprehensive medical history. Analysis of intraocular lymphocytes have also provided insight into the nature of chronic uveitis (Kaplan et al . , Archives of Qphthal ol. 102 (1984), pgs. 572-573). While the foregoing methods are useful, a need exists for a rapid, reliable procedure for diagnosing pars planitis and distinguishing that condition from other inflammatory conditions. Moreover, further understanding of molecular biological events associated with pars planitis will facilitate research into its underlying cause.
  • a novel pars planitis-specific circulating protein designated P- 36
  • the invention concerns preparations of P-36 and pars planitis-specific fragments thereof that are substantially devoid of contaminants of human origin.
  • the invention concerns cDNA molecules encoding P-36 and pars planitis-specific fragments thereof.
  • the invention concerns a DNA molecule represented by SEQ ID NO:l and a protein represented by SEQ ID NO:2.
  • the invention concerns an expression vector that contains a DNA sequence encoding P-36 or a pars planitis-specific fragment thereof and regulatory sequences capable of directing expression of such DNA sequence in a host cell transformed with such vector.
  • the invention concerns a host cell transformed with an expression vector that contains a DNA sequence encoding P-36 or a pars planitis-specific fragment thereof and a regulatory sequence capable of directing expression of such DNA sequences in such host cell.
  • the invention concerns oligonucleotide probes capable of detecting DNA or mRNA sequences that encode P-36 or pars planitis-specific fragments thereof .
  • the invention concerns a method of making P-36 or a pars planitis-specific fragment thereof by ( 1 ) cultivating in a nutrient culture medium cells transformed with an expression vector that contains a DNA sequence encoding P-36 or a pars planitis-specific fragment thereof and a regulatory sequence capable of directing expression of such DNA sequence in such cells, and ( 2 ) recovering the P-36 or pars planitis-specific fragment thereof.
  • the invention concerns polyclonal or monoclonal antibodies, antibody fragments or recombinant antibodies to P-36 or a pars planitis- specific fragment thereof.
  • the invention concerns a method of determining the presence of mRNA encoding P- 36 in a cell or tissue which involves the use of a labeled oligonucleotide probes complementary to P-36 mRNA in a Northern blotting or .in. situ hybridization procedure.
  • the invention concerns a method of diagnosing a patient for pars planitis which comprises determining the presence of P-36 in a biological fluid from such patient.
  • the present invention in its various aspects, provides a valuable tool to both clinicians and researchers.
  • the availability of P-36 protein and fragments thereof as well as antibodies and oligonucleotide probes enable the rapid and reliable diagnosis of the disease.
  • this valuable marker is expected to provide insight into the etiology and pathogenesis of the disorder and to facilitate drug screening and other therapeutic approaches .
  • P-36 A novel circulating protein with a molecular weight of 36 kDa (P-36) has been identified in the plasma of patients with pars planitis. Levels of P-36 are undetectable or very low in patients with other forms of uveitis, systemic inflammatory diseases (such as systemic lupus erythematosus and rheumatoid arthritis ) , diabetic retinopathy as well as in normal controls. Furthermore, plasma levels of P-36 have been found to correlate with disease activity in pars planitis.
  • a cDNA gene encoding P-36 has been isolated and cloned.
  • the nucleotide sequence of this cDNA gene and the deduced amino acid sequence of the P-36 protein are shown in SEQ ID NO:l and SEQ ID NO:2, respectively.
  • the cDNA sequence contains an open reading frame of 965 base pairs encoding a protein of 322 amino acids.
  • the cDNA sequence also contains a 5' untranslated region of 322 base pairs and a 3' untranslated region of 2693 base pairs. While the DNA sequence identified in SEQ ID NO:l was determined from the cDNA, the present invention encompasses not only cDNA, but synthetic DNA molecules and cloned genomic DNA oncoding P-36 as well.
  • Coding region nucleotide sequences have about 45-48% homology with human proteins such as endothelial leukocyte adhesion molecule (ELAM) , IFN-alpha, IFN- gamma, IL-2 and IL-6 suggesting a possible mediator role for P-36 in inflammation.
  • ELAM endothelial leukocyte adhesion molecule
  • the P-36 protein has been isolated from the plasma from pars planitis patients.
  • the amino-terminal amino acid sequence of the protein was determined by conventional sequencing techniques and this sequence was used for designing a set of degenerate c_- 32 P-labeled oligonucleotide probes.
  • a human spleen lambda phage cDNA library was screened with the labeled probes. Clones positive after tertiary screening were plaque purified and a clone was isolated that contained the full-length cDNA sequence for P-36. The clone may be obtained from the American Type
  • the cDNA encoding P-36 or a fragment thereof can be inserted into an expression vector for expression in prokaryotic or eukaryotic cells.
  • the P- 36 protein or a pars planitis specific fragment thereof can be expressed in E. coli under the control of the bacteriophage ⁇ P L promoter and the temperature sensitive repressor, cl857, in a plas id such as that described by A. R. Shatzman and M. Rosenberg in Methods in Enzvmolo ⁇ v. Vol. 152 (1987) pg. 661-673.
  • Other promoter systems may be used as well.
  • fusion proteins including not only promoter and operator sequences, but also sequences encoding a portion of the amino terminal peptide of the lac z gene product may be used to express P-36 as a fusion protein. Such fusion proteins may be useful for increasing the immunogenicity of P- 36 when preparing antibodies.
  • the expression plasmid may also contain elements to enhance translation efficiency such as a phage ⁇ . anti-termination site, a ⁇ _ ell, cro. or E.coli alK ribosome binding sites, and transcription terminator sites.
  • the expression plasmids may contain drug resistance markers to facilitate cell selection procedures, as well as multi-restriction site cloning banks for facilitating the insertion P-36 sequences into the vector.
  • Expression vec €ors containing P-36 sequences will be transformed into an appropriate E_;_ *-*- coli host and grown under conditions appropriate for the particular expression system utilized, according to methods well known to those of skill in the art.
  • a preferred embodiment may include the expression of P-36 in a prokaryotic expression vector which 0 contains a DNA signal sequence that will cause the protein to be secreted into the periplasmic space of the bacterial cell or into the culture media.
  • This mode of expression will allow P-36 to be purified from the culture medium or from lysed cells as a soluble protein using protein purification techniques known in the art such as those described in M.P. Deutscher, Guide to Protein Purifications, Methods in Enzymolo ⁇ v, Vol. 182 (1990). If P-36 is produced in an insoluble inclusion body like form, it will be recovered from an insoluble cell fraction isolated by methods such as those described by M. W. Pantoliano et al . , Biochemistry 30 (1991) pgs.
  • the insoluble pellet can then be solubilized in buffers containing chaotropic agents such as 6M guanidine hydrochloride or 8M urea.
  • the protein can be renatured by slowly diluting out the chaotropic agent.
  • the renatured protein can be concentrated then purified by methods well known in the art of protein purification such as ultrafiltration, and gel filtration and ion exchange chromatography.
  • Antibodies to P-36 may also be used to purify the recombinant protein by affinity chromatography. These purification procedures should be adaptable to allow isolation of P-36 on both small and large scale.
  • P-36 and pars planitis specific fragments thereof can also be expressed in mammalian cell lines.
  • mammalian host cells may be preferred, because mammalian expression systems generally provide recombinant eukaryotic proteins which are correctly folded and biologically active, unlike many of the prokaryotic expression systems which express eukaryotic proteins in inactive and often insoluble forms . Thus denaturation-renaturation steps may be avoided.
  • Expression of P-36 in mammalian cells may provide for correct post-translational modifications, such disulfide bond formation, glycosylation, phosphoryiation, oligomerization or specific proteolytic cleavage, any of which may be necessary for the protein's biological activity.
  • Suitable host cell lines for expressing eukaryotic proteins include both those which express foreign genes transiently such as the CHO cells (Chinese Hamster Ovary) transfected using calcium phosphate transfection procedures and COS cells (African green monkey cells) tranfected using the DEAE dextran mediated transfection methods; and those which express foreign genes constitutively such as the CHO constitutive transfections using dhfr (dihydrofolate reductase) amplification or bovine papilloma virus vectors.
  • Other cell lines known to be useful for expressing foreign eukaryotic proteins include HeLa cell lines and myeloma cells.
  • the features of these vectors include a prokaryotic origin of replication and a selection marker; a eukaryotic origin of replication such as the SV40 origin of replication; a transcription control region active in the particular host cell being used; a polyadenylation signal and site; and an intron sequence.
  • kits for utilizing inactivated lytic DNA viruses include those utilizing inactivated lytic DNA viruses such as the vaccinia viral vectors such as those described by G. L. Smith et al., Nature 302 (1983) pg. 490 and the insect Baculovirus expression system described by G. E. Smith et al., Molecular and Cell Biology 3 (1983) pg. 2156.
  • These lytic expression systems provide the advantage of having the expressed P-36 released into the cell medium in an active form.
  • P-36 protein and pars planitis specific fragments thereof expressed in mammalian cell systems can be readily purified by procedures known to those of skill in the art of protein purification. Gel filtration, ion exchange chromatography, immunoaffinity chromatography and combinations of these techniques may be used advantageously.
  • P-36 may be obtained in purified form substantially free of contaminants of human origin.
  • substantially free is meant that such contaminants are not present at a level sufficiently high to prevent the use for which the protein is to be put.
  • human derived materials such as a human serum albumin or other human proteins , may be intentionally added to P-36 preparations, and in these cases, those materials are not considered contaminants .
  • DNA sequences and methods described herein are useful not only for producing the P-36 protein, but also may be employed for producing variations and derivatives thereof, including pars planitis specific fragments thereof, all of which maintain one or more of the biological or biochemical properties of P-36.
  • variations and derivatives include proteins containing one or more amino acid substitutes, deletions or additions. These modifications can be made directly to the P-36 protein, but more easily are accomplished by altering the P-36 encoding DNA by procedures such as site-directed mutagenesis .
  • pars planitis specific fragment is meant a portion of the P-36 protein of sufficient length and sequence to distinguish it from comparable sized fragments of other human proteins .
  • P-36 includes intact P-36 protein as well as the variations, derivatives and pars planitis specific fragments thereof described above. It will also be understood that, when referring to nucleotide sequences encoding P-36 and its variations, derivatives and pars planitis specific fragments, such sequences include all modifications thereof permitted by the degeneracy of the genetic code.
  • the present invention also provides antibodies immunoreactive with P-36. These antibodies can be used in immunoassay and immunoaffinity purification procedures and may also have therapeutic value.
  • the antibodies may be polyclonal or monoclonal and made be raised by immunizing animals with native or recombinant P-36 protein. Antigenic fragments containing specific epitopes present on P-36 as well as P-36 fusion proteins may also be used as immunogens.
  • Polyclonal antibodies are made by injecting a mammal such as a rabbit, goat or sheep with purified or partially purified P-36 by procedures well known in the art, such as those described in E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, pages 53-139.
  • monoclonal antibodies are employed.
  • Monoclonal antibodies are prepared by injecting mice or rats with immunogenic preparations of P-36, removing the spleen from an animal which demonstrates an immune response to P-36, and fusing cells secreting antibodies with an appropriate myeloma cell line. Procedures for preparing and selecting hybridoma cell lines secreting antibodies for specific proteins are well known in the art and are described in references such as E. HarJow and D. Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, at 148-281, and J. W. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986.
  • Fab, Fab', Fv or F(ab' ) fragments of antibodies to P-36 which retain their specificities for P-36, can be prepared by methods known in the art, by digesting preparations of the antibodies with enzymes such as papain, or pepsin and by chemical reduction of disulfide bonds. Such antibody fragments may be advantageous in diagnostic assays, particularly assays done on plasma. Recombinant antibodies having specificity to P-36 protein, are also encompassed within the present invention. Such antibodies include chimeric antibodies, as described for example in U.S.
  • the P-36 protein and antibodies to this protein of the present invention are useful in the diagnosis of pars planitis.
  • the antibodies immunoreactive with P-36 can be used for screening plasma samples from patients for the presence of this protein as an indication of pars planitis . Screening may be done using one of the many types of immunoassays known to those of skill in the art of immunoassays. For example plasma samples can be resolved by electrophoresis on SDS polyacrylamide gels, then electroblotted onto nitrocellulose membranes and analyzed by the procedure known as Western blot analysis.
  • the membranes are fixed, washed in appropriate buffers, then incubated with P-36 antibodies, which will bind to any P-36 protein bands which are immobilized on the membrane.
  • P-36 antibody solution is rinsed away, the membranes are incubated with a second antibody which is labeled with an enzyme capable of being detected using a chromogenic substrate such as alkaline phosphatase or horseradish peroxidase in a sandwich assay procedure.
  • This second antibody will be immunoreactive for the P-36 antibody.
  • the P-36 is a mouse monoclonal antibody of the IgG subclass
  • the second antibody may be a rabbit or goat antibody which is specific for mouse IgG.
  • P-36 antibodies can be labeled directly with the enzyme.
  • An advantage of this type of immunoassay is that it will not only demonstrate the presence of P-36 in a patient, it will also provide information about the specific size, as measured by the protein's molecular weight, of any P-36 variants that might appear.
  • a diagnostic assay utilizing antibodies to P-36 may involve other types of heterogeneous or homogeneous immunoassays. It may be advantageous to use a radioimmunoassay (RIA) or an enzyme linked immunoabsorbent assay (ELISA) that will allow the screening of samples more rapidly than the Western blot assay.
  • RIA radioimmunoassay
  • ELISA enzyme linked immunoabsorbent assay
  • Antibodies to P-36 may be conjugated with a fluorescence marker such as fluorescein or rhodamine and used in immunoassays.
  • a fluorescence marker such as fluorescein or rhodamine
  • the use of fluorescent antibodies either in direct assays where the p-36 antibody is labeled or in a sandwich assay where a second antibody which binds to P-36 is labeled, will allow the localization of P-36 in tissue samples of patients to provide a prognosis of the development of pars planitis in a patient.
  • Antibodies to P-36 together with P-36 protein may be utilized in test kits for the qualitative and quantitative" determination of P-36 in serum or in tissue samples.
  • the test kit may contain reagents and instructions for developing Western blots of plasma protein samples for determining the presence or absence of P-36 in the plasma of a patient presenting an inflammatory condition of the eye.
  • the kit may contain reagents and instructions for conducting an ELISA or RIA on patient plasma samples .
  • Reagent kits adapted for use with automated clinical analyzers are also contemplated.
  • antibodies and appropriately labeled P-36 or fragments thereof may be incorporated with buffers and other reagents into diagnostic products for automated fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA), turbidimetric inhibition immunoassays (TINIA), particle enhanced turbidimetric inhibition immunoassays (PETINIA), enzyme mediated immunoassay technique (EMIT), nephelometric inhibition immunoassay (NIIA), substrate linked fluorescence immunoassay (SLFIA), apoenzyme reactivation immunoassay system (ARIS), and various known automated systems in which reagents and samples are deposited on porous paper or glass fiber matrices.
  • FPIA fluorescence polarization immunoassays
  • TINIA turbidimetric inhibition immunoassays
  • PETINIA particle enhanced turbidimetric inhibition immunoassays
  • oligonucleotide probes useful in Southern and Northern blot procedures for determining the presence of DNA and RNA sequences characteristic of P-36.
  • Such probes are DNA or RNA molecules containing from 5 to 1000 or more nucleotides.
  • the probes are of a length of from about 10 to 50 nucleotides, most preferably from about 10 to about 30 nucleotides. DNA probes are preferred.
  • the nucleotide sequence of the probes is substantially complementary to a sequence within the
  • P-36 cDNA preferably within the coding region and most preferably a sequence that is unique to P-36.
  • the probes are labeled for detection.
  • Various labels known in the art may be employed, examples of which include incorporation of radioactive atoms, such as phosphorous or sulfur, enzyme conjugation, fluorescent marker conjugation and the like.
  • the probes can also be used in situ hybridization assays for determining the presence and location of P-36 DNA or RNA sequences in cell nuclei or cytoplasm.
  • this technique may be performed by fixing tissue specimens or cell cultures on a slide, applying a solution of fluorescence labeled probe under hybridization conditions, rinsing off unbound probe and examining the slide by fluorescence microscopy.
  • the present invention thus provides novel and valuable compositions and methods for diagnosing and studying pars planitis.
  • the invention is further illustrated by the following examples, which are intended to illustrate but not limit the invention.
  • Plasma samples from patients with different types of uveitis and diabetic retinopathy were collected at the Washington University Eye Center. Patients were evaluated by history, clinical examination and diagnostic testing as previously described in Kaplan, et al., Archives of Ophthalmol. , 102 (1984), pgs. 572-573. Three groups of uveitis patients were included in this study: acute idiopathic anterior uveitis (HLA-B27+ and B27-), chronic idiopathic panuveitis and pars planitis (i.e., intermediate uveitis, chronic cyclitis and peripheral uveitis).
  • HLA-B27+ and B27- acute idiopathic anterior uveitis
  • chronic idiopathic panuveitis i.e., intermediate uveitis, chronic cyclitis and peripheral uveitis.
  • Plasma samples were also obtained from patients with proliferative diabetic retinopathy, status-post panretinal laser photocoagulation and from patients with rheumatoid arthritis and systemic Lupus erythematosus . Normal healthy subjects with no history of eye disease formed the control group. Plasma samples were used immediately or aliquoted and stored at -80°C.
  • the beads were washed twice with BBS and bound proteins were eluted by heating at 80°C in 0.25M Tris, 2% SDS, 10% glycerine pH 6.8.
  • SDS-PAGE was performed using a 10% slab gel as described in Laemmli, Nature, 227 , (1970), pg. 680. Before loading on the gel, samples were treated with 5% 2-mercaptoethanol and heated at 80°C for 10 minutes. Gels were fixed and silver stained as described in Merill, et al. , Science, 211, (1981) pgs. 1437-1438.
  • proteins from the polyacrylamide gels were transferred by electroblotting to a polyvinylidene diflouride (PVDF) membrane according to the procedures described in Matsudaira, J. Biol. Chem. , 262 (1987) pg. 10035 and LeGendre, Biotechnigues, ⁇ _ (1988) pg. 154.
  • PVDF polyvinylidene diflouride
  • a membrane slice containing the unique protein was cut out and filter-bound protein was subjected to NH 2 -terminal amino acid sequence analysis using an Applied Biosystems 477 Sequenator.
  • the probe was synthesized on an Applied Biosystems , Inc., DNA synthesizer (Model 380 A) . Recombinant phages were plated at a density of approximately 3 x 10 4 plaque forming units/150mm dish.
  • Duplicate plaque lifts on nitrocellulose filters were hybridized at 42°C overnight in 6 x SSC, lO M EDTA, 5 X Denhardt's solution, 0.1% SDS, 100 ug/ml denatured salmon sperm DNA, and the [ 32 P]-labeled oligonucleotide probe, having a specific activity of 2-4 x IC 6 cpm/ml (1 x SSC is 0.15 M NaCl, 0.015 M sodiuim citrate). Filters were washed twice in 2 x SSC, 0.1% SDS at room temperature and then twice in 1 x SSC, 0.1% SDS at 42°C for 15 minutes each time. Approximately 300,000 plaques from the library were screened.
  • the probe was end-labeled with gamma-[ 32 P]ATP and polynucleotide kinase.
  • Autoradiographs were prepared at -70°C on Kodak X-Omat AR film with Cronex intensification screens (Dupont Co., Wilmington, DE). Clones positive after tertiary screening were plaque purified by standard techniques.
  • Phage DNA was isolated and digested by EcoRI, electrophoresed on a 1% agarose gel, and the DNA was transferred to a nylon membrane (Oncor, Gaithersburg MD) overnight. Hybridization and washing were carried out as described for the plaque hybridization.
  • the cDNA insert was subcloned into the EcoRI site of pUC19 by standard techniques. DNA sequencing was performed by dideoxy-chain termination (Sanger et al., Proc. Natl. Acad. Sci. USA, 74 (1977), pg. 5463) using alkaline-denatured, double-stranded DNA templates (Chen, et al., DNA . NY . , 4_ (1985) pg. 165) and T7 polymerase as described in Tabor, et al . , Proc. Natl . Acad. Sci. USA, 84 (1987) pg. 4767 (U.S. Biochemical Corp., Cleveland OH).
  • Sequencing primers which were used at 15:1 molar ratio to DNA templates included forward and reverse Ml3 primers obtained from New England Biolabs, Beverly, MA, as well as oligonucleotides corresponding to regions of the sequenced cDNA. Both DNA strands were sequenced.
  • Nonspecific hybridization was removed by washing the filters two times at 62°C with buffer containing 1 x SSC and 0.1% SDS. Positive hybridization was identified by exposure to Kodak X-Omat AR films at -80°C overnight.
  • DNA fragments were transferred from the gel to the nylon membrane then hybridized with a P-36 cDNA probe, which was labeled with alpha[ 32 P]dCTP by random oligonucleotide priming as described above. Hybridization and high stringency washings were carried out using standard methods. Autoradiography was performed using Kodak X-Omat AR films at -80°C for 1-3 days.
  • GenBank release 68.0
  • EMBL release 27.0
  • NBRFPIR National Biomedical Research Foundation Protein Identification Resource
  • ACTCTTTGGC GCCACCGGCA CGAACTCTTC AGCAGTTGGT TCAACTGGTG GACTTTTTGG 120
  • GCT AAA TTT CAA AAG TGG GGC AAG ACA CTG GAA AGA AGT GAT AGA GGA 448 Ala Lys Phe Gin Lys Trp Gly Lys Thr Leu Glu Arg Ser Asp Arg Gly 30 35 40
  • AACTCATATG TCATGTCTAA AACTTGCCTA GAGGGGAATA GTATATGTTG AGCAAGTGCG 2938
  • Ser Ser lie Ala Ala Ser Lys Leu Thr Glu Lys Ala His Ser Pro Gin 115 120 125
  • Lys Asn Asn Tyr Tyr lie Ser Pro Ser lie Glu Thr Leu Gly Asn Lys 180 185 190
  • Cys Ser lie Tyr Glu Asn Cys Ser lie Lys Pro Glu Lys Gly Glu Gly 245 250 255 lie Asn Val Arg Cys Arg Val Thr Leu Tyr Ser Cys Phe Pro lie Asp 260 265 270
  • Lys Glu Thr Arg Lys Pro lie Lys Asn lie Thr His Pro Leu Leu Lys 275 280 285

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Abstract

Nouvelle protéine circulante spécifique de pars planitis, appelée P-36, présentant une masse moléculaire d'approximativement 36 kilodaltons. On a cloné le gène d'ADNc codant la P-36, et on a déterminé le nucléotide ansi que des séquences d'acides aminés déduites. L'invention concerne également des vecteurs et des systèmes d'expression de la P-36 recombinée. En outre, l'invention concerne des anticorps contre la P-36 ainsi que leur usage dans des immunodosages de la protéine, et un procédé de diagnostic de pars planitis chez un patient par détermination de la présence de la P-36 dans la circulation sanguine.
PCT/US1993/010825 1992-11-10 1993-11-10 Polypeptides specifiques de pars planitis WO1994011505A1 (fr)

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AU58963/94A AU5896394A (en) 1992-11-10 1993-11-10 Pars planitis specific polypeptides

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US97770992A 1992-11-10 1992-11-10
US07/977,709 1992-11-10

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5795872A (en) * 1995-09-19 1998-08-18 Pharmadigm, Inc. DNA construct for immunization
US5834235A (en) * 1996-06-21 1998-11-10 Health Research, Incorporated Inferferon-α-induced protein
US7531504B2 (en) * 1998-08-03 2009-05-12 University Of Maryland, Baltimore Pharmaceutical composition and method for inhibiting gastrointestinal inflammation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
E. HARLOW et al., "Antibodies, a Laboratory Manual", published 1988, by COLD SPRING HARBOR LABORATORY (COLD SPRING HARBOR), see pages 553-612. *
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Volume 32, No. 4, issued March 1991, N.S. BORA et al., "Identification of a Novel Circulating Protein (P-35) in Patients with Pars Planitis", page 940, Abstract No. 1335-73. *
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Volume 33, No. 4, issued 15 March 1992, J.B. FLEISCHMAN et al., "P-36. A Novel Circulating Protein; its Identification and Correlation with Active Pars Planitis", page 930, Abstract No. 1190-5. *
J. SAMBROOK et al., "Molecular Cloning, a Laboratory Manual", published 1989, by COLD SPRING HARBOR LABORATORY, see pages 11.1-11.61, 16.1-16.81 and 17.1-17.44. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5795872A (en) * 1995-09-19 1998-08-18 Pharmadigm, Inc. DNA construct for immunization
US5834235A (en) * 1996-06-21 1998-11-10 Health Research, Incorporated Inferferon-α-induced protein
US6060279A (en) * 1996-06-21 2000-05-09 Health Research, Incorporated Interferon-α-induced protein
US7531504B2 (en) * 1998-08-03 2009-05-12 University Of Maryland, Baltimore Pharmaceutical composition and method for inhibiting gastrointestinal inflammation

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