WO1994011505A1

WO1994011505A1 - Pars planitis specific polypeptides

Info

Publication number: WO1994011505A1
Application number: PCT/US1993/010825
Authority: WO
Inventors: Nalini S. Bora; Henry J. Kaplan
Original assignee: Washington University
Priority date: 1992-11-10
Filing date: 1993-11-10
Publication date: 1994-05-26
Also published as: AU5896394A

Abstract

A novel pars planitis-specific circulating protein, designated P-36, having a molecular mass of approximately 36 kilodaltons is disclosed. The cDNA gene encoding P-36 has been cloned, and the nucleotide and deduced amino acid sequences have been determined. Vectors and systems for expressing recombinant P-36 are described. Also described are antibodies to P-36 and their use in immunoassays for the protein. A method of diagnosing a patient for pars planitis by determining the presence of P-36 in the bloodstream is disclosed.

Description

PARS PLANITIS SPECIFIC POLYPEPTIDES

Background of the Invention

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.

Considerable confusion has existed in the field, because uveitis consists of many subsets of clinical diseases which are probably not causally related. 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 . Thus, it is a type of intermediate uveitis. It is a chronic inflammatory disease whose onset is generally insidious (Kraus-Macki , et al . , (eds. ) , Uveitis; Pathophysioloσv 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.

Summary of the Invention

In accordance with this invention a novel pars planitis-specific circulating protein, designated P- 36, has been identified and characterized. In one aspect, the invention concerns preparations of P-36 and pars planitis-specific fragments thereof that are substantially devoid of contaminants of human origin. In another aspect, the invention concerns cDNA molecules encoding P-36 and pars planitis-specific fragments thereof.

In a further aspect, the invention concerns a DNA molecule represented by SEQ ID NO:l and a protein represented by SEQ ID NO:2. In yet another aspect 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.

In an additional aspect, 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. In a further aspect, the invention concerns oligonucleotide probes capable of detecting DNA or mRNA sequences that encode P-36 or pars planitis-specific fragments thereof .

In yet another aspect, 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.

In another aspect, the invention concerns polyclonal or monoclonal antibodies, antibody fragments or recombinant antibodies to P-36 or a pars planitis- specific fragment thereof.

In a further aspect, 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.

In yet another aspect, 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.

Further aspects, advantages and uses of the compositions and methods encompassed within this invention will occur to those skilled in the art.

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. Moreover, 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 .

Detailed Description of the Invention

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. The deduced P-36 amino acid sequence lacks significant homology with known protein sequences. 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.

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_-³²P-labeled oligonucleotide probes.

Since the spleen plays an important role in various immune and inflammatory responses, 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

Culture Collection under accession no. ATCC 75407. Northern blot analysis of various tissue mRNA isolates was conducted using, as a probe, an c_-³P- labeled form of the cDNA insert described above. This analysis shows the presence of multiple mRNA species which may code for P-36 in spleen, eye and brain. In addition to 4.0 kb transcript that might code for 3.98 kb P-36 cDNA, three other transcripts of 6.0, 1.8 and 0.5 kb have been identified by Northern analysis. Analysis of the 3' untranslated region revealed the existence of putative polyadenylation signals that would generate the transcript of 1.8 kb. Whether these multiple transcripts represent the alternate selection of polyadenylation sites or encode distinct but related proteins, remains to be determined. Southern blot analysis of human geno ic DNA gave a simple hybridization pattern. This suggests that the P-36 gene is most likely present as a single copy per haploid genome and has a simple gene structure, i.e., is being encoded by a gene with few introns .

The cDNA encoding P-36 or a fragment thereof can be inserted into an expression vector for expression in prokaryotic or eukaryotic cells. For example, the P- 36 protein or a pars planitis specific fragment thereof can be expressed in E. coli under the control of the bacteriophage λ 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 Enzvmoloσv. Vol. 152 (1987) pg. 661-673. Other promoter systems may be used as well. DNA fragments from the lactose operon control region of E. coli. 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 λ. anti-termination site, a λ_ 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 Enzymoloσv, 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. 10117-10125. 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. Expression in 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. In addition to the transfection methods mentioned above it may be advantageous to introduce the DNA expression vectors containing the P-36 cDNA sequences into eukaryotic cells by electroporation. Versatile eukaryotic expression vectors, which have been designed for use in a wide range of eukaryotic cell lines, are well known in the art of eukaryotic cell expression. For example, such vectors are described in B. R. Cullen, Methods in Enzymology Vol. 152 (1987) pg. 684-704, and Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, Chapter 16. 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.

Other preferred expression vectors 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.

Whether expressed and purified from prokaryotic or eukaryotic recombinant cell systems, P-36 may be obtained in purified form substantially free of contaminants of human origin. By "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. Of course, for some uses, 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 .

The 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. Such 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 . By "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 . As used in the appended claims, it will be understood that the term "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. In a preferred embodiment 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. Patents 4,816,517 to Cabilly et al., and 4,816,397 to Boss, and CDR-grafted antibodies as described for example by Reich ann et al., Nature, 332 (1988), pgs. 323-327.

The P-36 protein and antibodies to this protein of the present invention are useful in the diagnosis of pars planitis. For example, 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. Following the electroblotting, 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. After the 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. For example, if 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. Alternatively 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.

Other embodiments of 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. Methods for preparing the reagents and reliable quantitative procedures for performing such assays are well known to those of skill in the art of immunoassay and are readily adapted to the detection and measurement of P-36 in plasma.

Antibodies to P-36 may be conjugated with a fluorescence marker such as fluorescein or rhodamine and used in immunoassays. 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. For example, 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. For example, 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. These various assay systems are in commercial use and are well known to those skilled in the art.

Also encompassed within this invention are 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. Preferably, 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.

In addition to the well-known Southern and Northern blot procedures, 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. In general, 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.

Example I

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).

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.

Example II Identification of P-36 and NH.-terminal Seguencing

250 ul of plasma samples were treated with polyethylene glycol-8000 (2.25% final concentration) at 4°C overnight. The resulting precipitate was resuspended in 100 μl of borate buffered saline (BBS) pH 8.0, incubated at 37°C for 30 minutes. The same was incubated with protein A beads at 4°C for 2-4 hours.

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. For amino terminal peptide sequence determination, 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, β_ (1988) pg. 154. A membrane slice containing the unique protein was cut out and filter-bound protein was subjected to NH₂-terminal amino acid sequence analysis using an Applied Biosystems 477 Sequenator. Example III Library Screening

A human spleen cDNA library (Clontech, Palo Alto, CA) in lambda gtlO was screened with [³²P]-labeled synthetic oligonucleotide probe. 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 10⁴ 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 [³²P]-labeled oligonucleotide probe, having a specific activity of 2-4 x IC⁶ 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-[³²P]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.

Example IV DNA Blot Hybridization

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.

Example V DNA Seguence Determination

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.

Example VI RNA Northern Blot Analysis

Total cellular RNA was isolated from human eyes by the guanidinium isothiocyanate/CsCl method of Chirgwin, et al., Biochemistry, 18 (1979) pg. 5294. Total RNA from human spleen and brain was purchased from Clontech, Palo Alto, CA. RNA was electrophoresed on a 1% agarose gel containing 2.2M formaldehyde. After transfer onto a nylon membrane (Oncor, Gaithersburg, MD) overnight, the RNA was hybridized (62°C) with an isolated insert of the P-36 cDNA labeled with alpha [³²P]dCTP by random hexanucleotide priming described in Feinberg, et al., Anal. Biochem. , 132 (1983) pg. 6. 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.

Example VII Southern Blot Analysis

Human genomic DNA was isolated from the nuclei of peripheral blood leukocytes by the method of Kunkel et al. (Kunkel, et al., Proc. Natl. Acad. Sci. USA, 74 (1977), pg. 1245). Approximately 15 ug of DNA was digested overnight with restriction enzymes (New England Biolabs, Beverly MA). According to the manufacturer's specifications. DNA fragments were size separated by size electrophoresis in 1% agarose gels prepared in 0.04M Tris acetate (pH 8.1), 0.025M sodium acetate, 0.002M EDTA at 100 V of constant voltage for 5 h. DNA fragments were transferred from the gel to the nylon membrane then hybridized with a P-36 cDNA probe, which was labeled with alpha[³²P]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.

Computer Analysis of Nucleic Acid and Protein Seguences

Nucleic acid and protein sequences were analyzed by the GenBank (release 68.0), EMBL (release 27.0), National Biomedical Research Foundation Protein Identification Resource (NBRFPIR, release 28.0) data base.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Bora, Nalini S. Kaplan, Henry J.

(ii) TITLE OF INVENTION: Pars Planitis Specific Polypeptides

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Rothwell, Figg, Ernst & Kurz

(B) STREET: 555 13th St NW Suite 700 East

(C) CITY: Washington

(D) STATE: D. C.

(E) COUNTRY: U.S.A.

(F) ZIP: 20004

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: 10-NOV-1992

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Figg, E. Anthony

(B) REGISTRATION NUMBER: 27,195

(C) REFERENCE/DOCKET NUMBER: 1886-101

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 783-6040

(B) TELEFAX: (202) 783-6031

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3980 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: Human Spleen cDNA Library

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 323..1291

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAATTCTTCC ACAATCGGCC AAAACAAACC AGCTTTTGGA GGCACAACCC GAAATACAGG 60

ACTCTTTGGC GCCACCGGCA CGAACTCTTC AGCAGTTGGT TCAACTGGTG GACTTTTTGG 120

CCAGAATAAT AATACGCTTA ATGTTGGTAC ACAAAATGTA CCACCTGTGA ACAATACCAC 180

CCAAAACACC CTTTTGGGTA CAACGGCAGT TCCTTCCCTA CAACAAGCTC CAGTAACTAA 240

TGAACAGCTT TTTTCCAAAA TATCAATCCC TAACTCTATT ACAAATCCAG TCAAAGCAAC 300

AACTTCAAAA GTGAACGCCG CC ATG AAA AGA CAA TTT GTC GAC TTC CCT AAG 352

Met Lys Arg Gin Phe Val Asp Phe Pro Lys 1 5 10

TAT AGA CTT GCC CCA AAG CCG TTA TTT GCT CCC TCT TCG AAT GGC GAT 400 Tyr Arg Leu Ala Pro Lys Pro Leu Phe Ala Pro Ser Ser Asn Gly Asp 15 20 25

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

AGC AGT ACC AGC AAT TCT ATT ACG GAT CCA GAA TCA AGC TAT CTA AAT 496 Ser Ser Thr Ser Asn Ser lie Thr Asp Pro Glu Ser Ser Tyr Leu Asn 45 50 55

TCA AAT GAC TTG TTG TTT GAT CCA GAT AGA AGA TAT TTG AAA CAT CTG 544 Ser Asn Asp Leu Leu Phe Asp Pro Asp Arg Arg Tyr Leu Lys His Leu 60 65 70

GTG ATT AAA AAT AAT AAG AAC TTA AAT GTC ATT AAC CAT AAT GAT GAT 592 Val lie Lys Asn Asn Lys Asn Leu Asn Val lie Asn His Asn Asp Asp 75 80 85 90

E SHEET GAA GCA AGC AAA GTT AAA TTA GTG ACG TTT ACA ACA GAA TCA GCT TCA 64 Glu Ala Ser Lys Val Lys Leu Val Thr Phe Thr Thr Glu Ser Ala Ser 95 100 105

AAA GAT GAC CAA GCC TCA TCA AGC ATT GCT GCT TCA AAA TTA ACT GAA 68 Lys Asp Asp Gin Ala Ser Ser Ser lie Ala Ala Ser Lys Leu Thr Glu 110 115 120

AAA GCA CAT TCT CCT CAG ACC GAC CTA AAA GAT GAT CAT GAT GAA AGC 736 Lys Ala His Ser Pro Gin Thr Asp Leu Lys Asp Asp His Asp Glu Ser 125 130 135

ACT CCT GAT CCT CAA TCG AAA GCT CCA AAC GGT TCC ACC TCT ATA CCA 784 Thr Pro Asp Pro Gin Ser Lys Ala Pro Asn Gly Ser Thr Ser lie Pro 140 145 150

ATG ATT GAG AAT GAA AAG ATT AGC AGC AAA GTT CCC GGC CTA TTG AGC 832 Met lie Glu Asn Glu Lys lie Ser Ser Lys Val Pro Gly Leu Leu Ser 155 160 165 170

AAC GAC GTT ACC TTT TTC AAG AAT AAC TAC TAC ATT TCA CCT TCC ATA 880 Asn Asp Val Thr Phe Phe Lys Asn Asn Tyr Tyr lie Ser Pro Ser lie 175 180 185

GAA ACG CTT GGC AAT AAG TCA TTA ATT GAA CTT CGT AAA ATA AAC AAC 928 Glu Thr Leu Gly Asn Lys Ser Leu lie Glu Leu Arg Lys lie Asn Asn 190 195 200

CTA GTC ATT GGT CAC AGA CAT TAT GGT AAA GTC GAG TTT CTG GAG CCC 976 Leu Val lie Gly His Arg His Tyr Gly Lys Val Glu Phe Leu Glu Pro 205 210 215

GTT GAT TTG TTG AAT ACT CCT TTG GAT ACT TTA TGC GGG GAT CTT GTC 1024 Val Asp Leu Leu Asn Thr Pro Leu Asp Thr Leu Cys Gly Asp Leu Val 220 225 230

ACC TTT GGA CCA AAA TCA TGT TCA ATA TAT GAA AAC TGT TCC ATA AAG 1072 Thr Phe Gly Pro Lys Ser Cys Ser lie Tyr Glu Asn Cys Ser lie Lys 235 240 245 250

CCA GAA AAG GGC GAA GGC ATT AAT GTA CGT TGT AGA GTG ACT TTA TAT 1120 Pro Glu Lys Gly Glu Gly lie Asn Val Arg Cys Arg Val Thr Leu Tyr 255 260 265

TCC TGT TTT CCT ATT GAC AAA GAA ACA AGG AAA CCT ATA AAG AAT ATA 1168 Ser Cys Phe Pro lie Asp Lys Glu Thr Arg Lys Pro lie Lys Asn lie 270 275 280

ACA CAT CCT CTA CTG AAA AGA AGT ATA GCC AAA CTA AAA GAA AAC CCA 1216 Thr His Pro Leu Leu Lys Arg Ser lie Ala Lys Leu Lys Glu Asn Pro 285 290 295

GTG TAC AAG TTT GAA AGC TAC GAC CCC GTA ACA GGC ACC TAT AGT TAC 1264 Val Tyr Lys Phe Glu Ser Tyr Asp Pro Val Thr Gly Thr Tyr Ser Tyr 300 305 310 ACC ATA GAT CAT CCA GTT TTA CCT TAAACCGGAA TAATTTTTGT AGAGAATCCT 1318 Thr lie Asp His Pro Val Leu Pro 315 320

TGTATCGTCT AAGTAGTCTA GATGTTCAGC TGATAGATTT TCGTTGTATT GTATATATAA 1378

TAATTGTCGG AAAACAAAAA TACTTAATTA TAATTGTGTG ACCGAAAATG CCTGATCAAC 1438

AGCCATGGCA CATTTGAATG GGATTTTGAG GAGACAAAAA TGAAGAGGTT CTATAACCTT 1498

TGTAGAAGAA CATCGACCTC TTTTCAATGA AGATTGGTGG CATCGCCAGA TGCAGAAAGT 1558

TTGCCTCATG AAATAAAAAT GAACAGACCA TGAAGGCTAA AGAAAGAAAA AAATAAAATG 1618

CCATCTCCTA GAATCGAACC AGGGTTTCAT CGGCCACAAC GATGTGTACT AACCACTATA 1678

CTAAGATGGC AAACAACTGT GAAATATTTG GTTACATCTG CACATCTGGT GGAATAAATA 1738

ATATGTACTC TTTGCTTTTT TATGTTAAAC CTACAAGTGG TGACTGTAAA GAAGCATTAC 1798

AACGTAGAAC TGATAAAGGA GAGTAGTTAC ATAAGCTTTC CGTAATGGTG AATTTATAGC 1858

AGTTTTCTTC TCGATGAAAG AAAGGGAAAG AACTAAATAT ACTCGAATGC TTGTACCACT 1918

CCATTTCCCC ATTTATCACA TTTAAAGTTA CGAGTAAAAA AGTGACCGAT ATAGAATGTC 1978

TGATGAAAGT GAGATATATG TGGGTAATTA GATAATTGTT GGGATTCCAT TGTTGATAAA 2038

GGCTATAATA TTAGGTATAC AGAATATACT AGAAGTTCTC CTCGAGGATA TAGGAATCCT 2098

CAAAATGGAA TCTATATTTC TACATACTAA TATTACGATT ATTCCTCATT CCGTTTTATA 2158

TGTTTATATT CATTGATCCT ATTACATTAT CAATCCTTGC GTTTCAGCTT CCTCTAACAT 2218

CGATGACAGC TTCTCATAAC TTATGTCATC ATATTAACAC TGAATATGAT AATATATTGA 2278

TAATATAACT ATTAGTTATA GACGATAGTG GATTTTTATT CCAACATACC ACCCATAAAG 2338

TAATAGATCT AATGAATCCA TTTGTTTGTT TATAGTTTAA ATGTTTTTAT CGGAAGAGGT 2398

TTTGTCATCA CATCAGCAAT GTTCTTCTTG GTCTCGATGT AGTATACGTA TACATTATTA 2458

CCTGATACTT CATCTCTAAG TCTCATTGCC TTTGTGAAAA AAAATCTGTT TCTAAATTTC 2518

TCTTCATTTG TAGACTTAAT TATACTGATC GTTGATCTAC TATCAGTAAG TAAGCCTTTA 2578

ATAATTGGTT TCTTGTTAAG TTCTTGCACA AGGTGACTGA GGTTATTCAA TAGCGGAATA 2638

GCTTCACTGA CTGCGTGTAT TTCTGCTTCT GTAGTTGAAG TGCATGTTAA CGAAGCCTTT 2698

GTCGACTTTC CTCCAATCAC TTTTCCGTTG AGTAGGAAAA TGTTACCAAT TTGTGACTTG 2758

TAATATGGTT GGTTACCATA TGAAGCATCG CTTATTGCGA CTAGTTTATT ATCTGGCTTG 2818

GTAGGTTTGT TTTTGTGCCA TATTAATTGT TTATCTCTAG TGTCCCACAT GAATTGTATT 2878

AACTCATATG TCATGTCTAA AACTTGCCTA GAGGGGAATA GTATATGTTG AGCAAGTGCG 2938

TTGATGTAGT ATAGTAAGCC AAATCTAAAT TTATATCCAA CATATGAAGC TAGACCAATC 2998

SUBSTITUTE SHEET AACTTTTGCA TTTCATGTAC CTTCTCTTTG TATTCATCTT CATCTATTTC TAGTTCATCC 3058

TGGTCTATAT AAAGACCTGG TTGACCTGGA GCGCGAAGTT TTCTTCCTTT TGGATTCAAA 3118

GGTACGTTTA ATTTGGGTAT TTTCTCAGTT AATGAGTTTT CCATACCTAA TTTCATGTAT 3178

TTACCTCTTT GATATTTGAT TTCTAAGCCA AGTATGTCGT ACTGAATTTC GTTATCACTT 3238

TCACCCAGAT TTATTATCTT TGTATCGTAT TGTTTCTTGA GTGTTGTTAT GATTTTCTTA 3298

TTTGCATTTA AGTCTTTGCT GAACAATATC ATATCATCAA CGAATAAGCA AATTGTTACT 3358

GGACTATTCT TAAATACGCA TGACCATCCA CGAACTTCTT CCATACCACA CTGTTTTATC 3418

AGGTATGATT TGATAGTTTC GTACCAGTTC GCTCCACTTT GTTTCAATCC ATAAAGTGAT 3478

TTCTTCAAAC GTATCAACTT ATCATTCATT CCTAAATGTG GTGGAGGTCT TATGTATAAT 3538

TCTTCTTTGA TGTCTGCATA CAAATATGCC GAAGATATGT CTAATTGTGT AATATAGTAG 3598

TTATTGTCTA ATGCAAGTGA CAGGGATGTC ATTAATGCAT AGTGATGTAC GGTATTGGAT 3658

TGCATGCCTG AGTCGTAAGT GTCAGGATGC TGAATATCAC CTCTTGCAAC AAATCTAGCT 3718

TTATGAGTAC CGTCACGTTT CCTGTTGAAG ATAAACATTG AATTTATTAC TCTTTTAGGG 3778

TCTATTTCTT TTCTGTCATA ATAAATGTCA GTGTCCCAAG TATTCATTTT CAATAGTTGG 3838

TTGACTTCTT TGTGGTATGC TTCGATATAT TTTTCCTTTT CTTTAATATC TTTATTATAG 3898

GTGATTGCCT CATCGTATCT TAAGGTTGTC CGTATTGGTT TGATTGATTT TACTGCTTTT 3958

ACAGCTGCAA TCAGGTGAAT TC 3980

(2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 322 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Arg Gin Phe Val Asp Phe Pro Lys Tyr Arg Leu Ala Pro Lys 1 5 10 15

Pro Leu Phe Ala Pro Ser Ser Asn Gly Asp Ala Lys Phe Gin Lys Trp 20 25 30

Gly Lys Thr Leu Glu Arg Ser Asp Arg Gly Ser Ser Thr Ser Asn Ser 35 40 45 lie Thr Asp Pro Glu Ser Ser Tyr Leu Asn Ser Asn Asp Leu Leu Phe 50 55 60

Asp Pro Asp Arg Arg Tyr Leu Lys His Leu Val lie Lys Asn Asn Lys 65 70 75 80

Asn Leu Asn Val lie Asn His Asn Asp Asp Glu Ala Ser Lys Val Lys 85 90 95

Leu Val Thr Phe Thr Thr Glu Ser Ala Ser Lys Asp Asp Gin Ala Ser 100 105 110

Ser Ser lie Ala Ala Ser Lys Leu Thr Glu Lys Ala His Ser Pro Gin 115 120 125

Thr Asp Leu Lys Asp Asp His Asp Glu Ser Thr Pro Asp Pro Gin Ser 130 135 140

Lys Ala Pro Asn Gly Ser Thr Ser lie Pro Met lie Glu Asn Glu Lys 145 150 155 160 lie Ser Ser Lys Val Pro Gly Leu Leu Ser Asn Asp Val Thr Phe Phe 165 170 175

Lys Asn Asn Tyr Tyr lie Ser Pro Ser lie Glu Thr Leu Gly Asn Lys 180 185 190

Ser Leu lie Glu Leu Arg Lys lie Asn Asn Leu Val lie Gly His Arg 195 200 205

His Tyr Gly Lys Val Glu Phe Leu Glu Pro Val Asp Leu Leu Asn Thr 210 215 220

Pro Leu Asp Thr Leu Cys Gly Asp Leu Val Thr Phe Gly Pro Lys Ser 225 230 235 240

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

Arg Ser lie Ala Lys Leu Lys Glu Asn Pro Val Tyr Lys Phe Glu Ser 290 295 300

Tyr Asp Pro Val Thr Gly Thr Tyr Ser Tyr Thr lie Asp His Pro Val 305 310 315 320

Leu Pro

Claims

1. A P-36 protein preparation substantially free cf contaminants of human origin.

2. The protein of claim 1, having the amino acid sequence described in SEQ ID NO:2.

3. A cDNA molecule comprising a nucleotide sequence encoding P-36.

4. The cDNA molecule of claim 3, having the nucleotide sequence described in SEQ ID N0:1.

5. The cDNA molecule of claim 3, comprising nucleotide residues 323 through 1291 of SEQ ID N0:1.

6. A synthetic DNA molecule encoding P-36.

7. A genomic DNA clone encoding P-36.

8. An expression vector comprising a DNA sequence encoding P-36 and a regulatory sequence capable of directing expression of such DNA sequence in a host cell transformed with such expression vector.

9. The expression vector of claim 8, wherein said DNA sequence has the nucleotide sequence described in SEQ ID N0:1.

10. The expression vector of claim 8, wherein said DNA sequence comprises nucleotide residues 323 through 1291 of SEQ ID NO:l.

11. The expression vector of claim 8, wherein the regulatory sequence is one that is capable of directing expression in a prokaryotic host cell.

12. The expression vector of claim 11, wherein the host cell is E.coli.

13. A P-36-producing prokaryotic host cell transformed with the expression vector of claim 11.

14. The expression vector of claim 8, wherein the regulatory sequence is one that is capable of directing expression in a eukaryotic host cell.

15. The expression vector of claim 14, wherein the eukaryotic host cell is a mammalian cell.

16. The expression vector of claim 15, wherein the mammalian cell is a Chinese hamster ovary cell.

17. The expression vector of claim 15, wherein the mammalian cell is a COS cell.

18. The expression vector of claim 15, wherein the mammalian cell is a HeLa cell or a myeloma cell.

19. A P-36-producing mammalian cell line transformed with the vector of claim 15.

20. A method for producing P-36, which comprises (1) cultivating in a nutrient culture medium cells transformed with an expression vector that contains a DNA sequence encoding P-36 and a regulatory sequence capable of directing expression of such DNA sequence in such cells and (2) recovering the P-36.

21. The method of claim 20, wherein the cells are prokaryotic cells.

22. The method of claim 21, wherein the cells are E.coli.

23. The method of claim 20, wherein the cells are eukaryotic.

24. The method of claim 23, wherein the cells are mammalian.

25. The method of claim 24, wherein the mammalian cells are selected from Chinese hamster ovary cells, COS cells, HeLa cells and myeloma cells.

26. An antibody to P-36.

27. The antibody of claim 26, wherein the antibody is a polyclonal antibody.

28. The antibody of claim 26, wherein the antibody is a monoclonal antibody.

29. The antibody of claim 26, wherein the antibody is a recombinant antibody.

30. An immunoassay method for the determination of P-36 in a sample, which comprises contacting the sample with an antibody to P-36 and determining the presence or absence of P-36 by the extent of binding of such antibody to P-36 in the sample.

31. The method of claim 30, wherein the immunoassay is a Western blot assay.

32. The method of claim 30, wherein the immunoassay is an enzyme linked immunoabsorbent assay.

33. The method of claim 30, wherein the immunoassay is a radioi munoassay.

34. A reagent kit for the immunoassay of a sample for P-36, which comprises antibody to P-36 and labeled P-36 or a labeled fragment thereof which is capable of reacting with such antibody.

35. A method of diagnosing a patient for pars planitis, which comprises determining the presence of P-36 in the bloodstream of such patient.

36. An oligonucleotide probe having a sequence substantially complementary to a sequence within P-36 cDNA.

37. The oligonucleotide probe of claim 36, wherein the sequence is substantially complementary to a sequence within the coding region of P-36 cDNA.

38. The oligonucleotide probe of claim 36, wherein the sequence is unique to the P-36 cDNA.

39. The oligonucleotide probe of claim 36, which is an oligodeoxyribonucleotide.

40. The oligonucleotide probe of claim 36, which is an oligoribonucleotide.

41. The oligonucleotide probe of claim 38, which is an oligodeoxyribonucleotide having a length of from about 10 to about 30 nucleotides and having a detectable label.