WO1991008289A1 - Sperm specific nuclear autoantigenic protein - Google Patents

Abstract

Disclosed is a DNA sequence which codes for Sperm Nuclear Autoantigenic Protein (SNAP) or a fragment thereof. Essentially pure SNAP is also disclosed. The DNA sequence which codes for human SNAP and the DNA sequence which codes for rabbit SNAP are specifically disclosed. SNAP is useful as an immunocontraceptive agent and in the diagnosis of autoimmune infertility.

Description

SPERM SPECIFIC NUCLEAR AUTOANTIGENIC PROTEIN

Field of the Invention

This invention relates to the identification of a sperm specific nuclear protein which is an autoantigen (SNAP) , and DNΛ sequences coding for the same. Background of the Invention

Λutoantigens are tissue components of an organism to which that organism directs an immune response. The condition which results from such a self-directed immune response is known as autoimmunity (or "autoallergy") . Proteins in or on sperm are known to be potent autoantigens, and autoimmunity to such proteins is believed a significant cause of infertility.

Sperm is formed through a dramatic reorganization of cell architecture known as spermatogenesis. During this process, events such as the formation of the acrosomal vesicle, the elaboration of the flagellum, the condensation of the nucleus, and the removal of excess cytoplasm are all required for the formation of a viable spermatozoon. Coincident with these events is an extensive reorganization of spermatozoan membrane components to form highly specialized, regionally specific, functional domains (D. Friend et al., J. Cell Biol. 7 , 561 (1977) ; M. O'Rand and L. Romrell, Devel. Biol. 75, 431 (1980) ; M. O'Rand and L. Ro rell, Develop. Biol. 84, 322 (1981) ; D. Myles et al., J. Cell Biol. 98, 1905 (1984); P. Primakoff et al. , J. Cell Biol. 101, 2239 (1985) ; and J. Welch and M. O'Rand, Develop. Bio3. 109 , 411 (1985)) . In the plasma membrane intramembranous particles are reorganized and boundaries appear, such as the regular serrations between acrosomal and postacrosomal regions (J. Koehler, J. Ultrast. Res. 33, 598 (1970)), the posterior ring between postacrosomal and tail regions and the annuluε between mitochondrial and principal piece regions (D. Friend and D. Fawcett, J. Cell Biol. 63 , 641 (1974)) . The nuclear envelope also undergoes extensive reorganization with the patching of nuclear pores followed by their movement to the posterior nuclear pole and almost complete removal from the nuclear envelope (D. Fawcett and H. Che es, Tissue and Cell JL1, 147 (1979) ) .

Spermatogenesis may also be characterized by the appearance of testis specific proteins which function in the mature spermatozoon (M. O'Rand and L. Romrell, Devel. Biol. 55 , 347 (1977)). One such sperm specific protein which first appears in pachytene spermatocytes is the plasma membrane autoantigen rabbit sperm membrane autoantigen, or "RSA." M. O'Rand and L. Romrell, Develop. Biol. 84 , 322 (1981) (also called "RSA-1") . RSA is concentrated in the postacrosomal region of the mature spermatozoon (N. Esaguy et al., Gamete Res. 19 , 387 (1988)) and functions as a lectin-like protein to bind sperm to the zona pellucida (M. O'Rand et al. , Develop. Biol. 129. 231 (1988)) .

Summary of the Invention

In our studies of RSA, we unexpectedly found an i munologically cross reactive protein present in the sperm's postacrosomal region, but which appears to be initially associated with the nuclear membrane during spermatogenesis.

This protein contains a C-terminal nuclear translocation signal, is sperm and testis specific and reacts strongly with autoantibodies to rabbit spermatozoa. This protein is called sperm specific nuclear autoantigenic protein, or "SNAP."

Interestingly, SNAP shows considerable ho ology to the histone-binding protein, N1/N2, from Xenopus laevis. Therefore, SNAP appears to be a novel testis and sperm specific nuclear protein which has evolved from an amphibian precursor sequence. Accordingly, a first aspect of the present invention is an essentially pure DNA sequence which codes for SNAP or a fragment thereof. Λ more particular aspect of this invention is an essentially pure DNA sequence which codes for a protein which bind antibodies which bind to SNAP and is capable of hybridizing to the DNA sequence of either Figure 1 or Figure 2 herein.

A second aspect of the present invention is a gene transfer vector containing a DNA sequence which codes for SNAP or a fragment thereof. A third aspect of the present invention is a microbial host transformed by a gene transfer vector containing a DNA sequence which codes for SNAP or a fragment thereof.

A fourth aspect of the present invention is a process for the preparation of SNAP comprising culturing the aforesaid microbial host transformed by a gene transfer vector containing a DNA sequence which codes for SNAP or a fragment thereof under conditions suitable for the expression of SNAP and recovering SNAP from the cultured host. Λ fifth aspect of the present invention is essentially pure SNAP (e.g., human SNAP, rabbit SNAP) .

A sixth aspect of the present invention is an immunocontraceptive method comprising administering an animal subject an immunogen selected from the group consisting of SNAP, SNAP fragments, and derivatives thereof which bind selection antibodies which bind to SNAP in an amount effective to reduce the fertility of the subject.

A seventh aspect of the present invention is a method of diagnosing autoimmune infertility in a human subject. The method comprises the step of, first, contacting an immune sera sample from the subject with an antigen, the antigen selected from the group consisting of SNAP, SNAP fragments, and derivatives thereof which bind selection antibodies which bind to SNAP. The next step is to detect the presence of antibodies from the immune sera sample bound to the antigen, the presence of antibodies bound to the antigen suggesting the subject is afflicted with autoimmune infertility.

Brief Description of the Drawings The foregoing and other aspects and features of the present invention are explained in detail in the specification herein, in which:

Figures 1A to ID provide the complete rabbit SNAP DNA coding sequence (deduced amino acid residues are given) ;

Figures 2A to 2B provide the complete human SNAP cDNA sequence; and Figure 3 illustrates a serial dilution ELISA of serum from a vasectomized male with SNAP.

Detailed Description of the Invention For the purposes herein, SNAP is defined as a protein or polypeptide which is substantially homologous with the amino acid sequences coded for by the DNA sequence depicted in Figures 1 or 2, or fragments thereof, excluding any protein or polypeptide which exhibits substantially the same or lesser homology to the sequences coded for by the DNA sequences of Figures 1 or 2 than does the Xenopus laevis histone-binding protein, N1/N2. Ordinarily SNAP polypeptides will be about from 50 to 100 percent homologous to the proteins coded for by the Figures 1 and 2 sequences, preferably 80 to 100 percent homologous, and they will exhibit at least some biological activity in common with these sequences. Biological activity shall include, but is not limited to, cross-reactivity with anti-SNAP antibodies raised against SNAP from natural (i.e., nonrecombinant) sources. Preferably the derivatives will cross-react with antibodies which bind to to sperm. Thus, DNA sequences which code for SNAP include DNA sequences which encode such homologous proteins, which DNA sequences are capable of hybridizing with the DNA of Figures 1 or 2.

Homology is herein determined by optimizing residue matches by introducing gaps as required but without considering conservative substitutions as matches. This definition is intended to include natural allelic variations in SNAP sequences.

SNAP includes the SNAP of animals other than rabbits and humans, e.g. bovine, porcine, ovine, murine, and avian

SNAP. Preferably the SNAP is mammalian SNAP. PreSNΛP is a species of SNAP included within the foregoing definition. It is characterized by the presence in the molecule of a signal polypeptide, specifically a nuclear translocation signal, which serves to post-translationally direct the protein to a site inside or outside of the cell. Generally, the signal polypeptide (which will not have SNAP activity in its own right) is proteolytically cleaved from a residual protein having SNAP activity as part of the secretory process in which the protein is transported to the host cell nucleus, periplas , or culture medium. The signal peptide may be microbial or mammalian, but it preferably is mammalian.

1. Definitions.

In order to simplify the disclosure provided herein certain frequently occurring words, methods and phrases are referenced by shorthand expressions. In accordance with convention, nucleotide bases are referred to herein as follows:

A=λdenine G=Guanine

C=Cytosine T=Thymine

Similarly, in accordance with convention, amino acid residues are abbreviated as follows:

Λla=Alanine Leu=Leucine

Arg=Λrginine Lys=Lysine

Asn=Λsparagine Met=Methionine

Λsp=Aspartic acid Phe=Phenylalanine Cys=Cysteine Pro=Proline

Gln=Glutamine Ser=Serine

Glu=Glutamic acid Thr=Threonine Gly=Glycine Trp=Tryptophan His=Histidine Tyr=Tyrosine Ile=Isoleucine Val=Valine

The term "antigenic equivalents," as used herein, refers to proteins or peptides which bind to an antibody which binds to the protein or peptide with which equivalency is sought to be established. Antibodies which are used to select such antigenic equivalents are referred to as "selection antibodies" herein. "Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific is called a restriction site. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are established by the enzyme suppliers. Appropriate buffers and substrate amounts for particular restriction enzymes are also specified by the manufacturer. "Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the digest by polyacrylamide gel electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the DNA from the gel. This procedure is known generally. For example, see R. Lawn et al., Nucleic Acids Res. 9_, 6103 (1981), and D. Goeddel et al., Nucleic Acids Res. 8_., 4057 (1980) .

"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integrant.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. See T. Maniatis et al., Molecular Cloning: A Laboratory Manual , 146 (1982) .

2. SNAP Derivatives

The production of derivatives of proteins or protein fragments produced from DNA coding sequences is well known. See, e.g. , U.S. Patent No. 4,761,371 to Bell et al. at Col. 4 line 27 to Col. 5 line 56. Hence, the discussion which follows is intended as an introduction to this field, and does not represent the full level of skill in this art.

In general, derivatives of SNAP and SNAP fragments bind selection antibodies which bind to SNAP and have (a) one or more amino acid residues changed, (b) one or more amino acid residues deleted, or (c) one or more amino acid residues changed and one or more amino acid residues deleted-

Derivatives of SNAP include amino acid sequence mutants, glycosylation variants and covalent or aggregative conjugates with other chemical moieties. Covalent derivatives are prepared by linkage of functionalities to groups which are found in the SNAP amino acid side chains or at the^'N- or C- termini, by means known in the art. These derivatives may, for example, include: aliphatic esters or amides of the carboxyl terminus or residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or a ino-group containing residues, e.g. lysine or arginine. Λcyl groups are selected from the group of alkyl-moieties (including C3 to C18 normal alkyl) , thereby forming alkanoyl aroyl species. A major group of derivatives are covalent conjugates of SNAP or its fragments with other proteins or polypeptides. These derivatives are synthesized in recombinant culture as N- or C- terminal fusions or by the use of difunctional agents known per se for use in cross-linking proteins to insoluble matrices through reactive side groups.

Preferred SNAP derivatization sites with cross-linking agents are at cysteine and lysine residues. Preferred agents are M-Maleimidobenzoyl succinimide ester and N-hydroxysuccinimide. Covalent or aggregative derivatives are useful as immunogens, reagents in immunoassay or for affinity purification of SNAP. For example, SNAP is insolubilized by covalent bonding to cyanogen bromide-activated Sepharose by methods known per se or adsorbed to polyolefin surfaces (with or without glutaraldehyde cross-linking) for use in the assay or purification of anti-SNAP antibodies. SNAP also is labelled with a detectable group, e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in diagnostic assays.

Mutant SNAP derivatives include the predetermined, i.e. site specific, mutations of SNAP or its fragments. Mutant SNAP is defined as a polypeptide otherwise falling within the homology definition for SNAP as set forth herein but which has an amino acid sequence different from that of SNAP as found in nature, whether by way of deletion, substitution or insertion.

While the mutation sites are predetermined, it is unnecessary that the mutation per se be predetermined. For example, in order to optimize the performance of mutants at a given residue position, random mutagenesis may be conducted at the target codon and the expressed SNAP mutants screened for the desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis.

SNAP mutagenesis is conducted by making amino acid insertions, usually on the order of about from 1 to 10 amino acid residues, or deletions of about from 1 to 30 residues. Substitutions, deletions, insertions or any subcombination may be combined to arrive at a final construct. Insertions include amino or carboxyl-terminal fusions. Obviously, the mutationr. in the DNA must not place coding sequences out of reading frame and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.

Not all mutations in the DNA which encode SNAP will be expressed in the final product. For example, a major class of DNA substitution mutations are those in which a different secretory leader or signal has been substituted for the native human secretory leader, either by deletions within the leader sequence or by substitutions, wherein most or all of the native leader is exchanged for a leader more likely to be recognized by the intended host. However, the human secretory leader will be recognized by hosts other than human cell lines, most likely in cell culture of higher eukaryotic cells. When the secretory leader is "recognized" by the host, the fusion protein consisting of SNAP and the leader ordinarily is cleaved at the leader-mature SNAP peptide bond in the events that lead to secretion of SNAP or its insertion into the cell membrane. Thus, even though a mutant preSNΛP is synthesized as an intermediate, the resulting SNAP will be mature.

Another major class of DNA mutants that are not expressed as SNAP derivatives are nucleotide substitutions made to enhance expression, primarily to avoid amino terminal loops in the transcribed mRNA (see EPO Patent Application No. 75,444A) or to provide codons that are more readily transcribed by the selected host, e.g. the well-known E. coli preference codons for E. coli expression.

Compositions comprising SNAP may include predetermined amounts of proteins from the cell or organism that served as the source of DNA encoding the SNAP, proteins from other than the SNAP source cells or organisms, and synthetic polypeptides such as poly-L-lysine. Recombinant SNAP which is expressed in allogeneic hosts of course will be expressed completely free of gene source proteins. For example, the expression of human SNAP in Chinese Hamster Ovary (CHO) or other higher mammalian cells results in a composition where the SNAP is not only free of human proteins but the SNAP in the culture is not denatured.

3. SNAP Genetic Engineering techniques. Like the formation of derivative molecules, the production of proteins and protein fragments by genetic engineering is well known. See, e.g. , U.S. Patent No. 4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65. The discussion which follows is accordingly intended as an overview of this field, and is not intended to reflect the full state of the art.

DNA which encodes SNAP is obtained by chemical synthesis, by screening reverse transcripts of mRNA from testes cells or cell line cultures, or by screening genomic libraries from any cells.

This DNA is covalently labelled with a detectable substance such as a fluorescent group, a radioactive atom or a chemiluminescent group by methods known per se. It is then used in conventional hybridization assays. Such assays are employed in identifying SNAP vectors and transformants as described in the Examples below.

SNAP is synthesized in host cells transformed with vectors containing DNA encoding SNAP. A vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding SNAP and/or to express DNA which encodes SNAP. An expression vector is a replicable DNA construct in which a DNA sequence encoding SNAP is operably linked to suitable control sequences capable of effecting the expression of SNAP in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

Vectors comprise plasmids, viruses (including phage) , and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination) . The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "vector" is generic to "phage", but bacteriophage Lambda GT11 is the preferred vector at present. However, all other forms of vectors which serve an equivalent function are suitable for use herein. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. Transformed host cells are cells which have been transformed or transfected with SNAP vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express SNAP, but host cells transformed for purposes of cloning or amplifying SNAP DNA do not need to express SNAP. Expressed SNAP will be deposited around the nuclear membrane, in the cell membrane or secreted into the culture supernatant, depending upon the SNAP DNA selected.

DNA regions are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a riboso e binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading phase.

Suitable host cells are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example Escherichia coli (E. col.i ) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). Pseudomonas species, Bacillus species, and Serratia Marcesans are also suitable.

Prokaryotic host-vector systems are desireable for the expression of SNAP fragments that do not require extensive proteolytic and disulfide processing. A broad variety of suitable microbial vectors are available. Generally, a microbial vector will contain an origin of replication recognized by the intended host, a promoter which will function in the host and a phenotypic selection gene, for example a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement. Similar constructs will be manufactured for other hosts. E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, et al., Gene 2 , 95 (1977)) . pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host. Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (3978) ; and Goeddel et al. , Nature 281, 544 (1979)), a tryptophan (trp) promoter system (Goeddel et al. , Nucleic Acids Res. 8_., 4057 (1980) and EPO App. Pu l. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983)). While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many have been published, enabling a skilled worker operably to ligate them to DNA encoding SNAP in plasmid or viral vectors (Siebenlist et al., Cell 20, 269 (1980)). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA encoding the SNAP, i.e., they are positioned so as to promote transcription of SNAP messenger RNA from the DNA.

Eukaryotic microbes such as yeast cultures may be transformed with SNAP-encoding vectors. see, e.g. , U.S. Patent No. 4,745,057. Saccharo yces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS) , a promoter, DNA encoding SNAP, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al. , Nature 282, 39 (1979) ; Kingsman et al., Gene 1_, 141 (1979) ; Tschemper et al., Gene 10_f 157 (1980)) . This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 8J5, 12 (1977)) . The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al . , J. Adv. Enzyme Reg. 7, 149 (1968) ; and Holland et al. , Biochemistry 17, 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Pub_n. No. 73,657.

Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose utilization. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3 ' of the SNAP coding sequences to provide polyadenylation and termination of the mRNA. Cultures of cells derived from multicellular organisms are a desireable host for recombinant SNAP synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred. Propagation of such cells in cell culture has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used) , a polyadenylation site, and a transcriptional termination sequence.

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40) . See, e.g. , U.S. Patent No. 4,599,308. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273, 113 (1978)) . Further, it is also possible, and may be desirable, to utilize the human genomic SNAP promoter, control and/or signal sequences, provided such control sequences are compatible with the host cell chosen.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV 40 or other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the SNAP DNA. An example of a suitable selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. Such markers are proteins, generally enzymes that enable the identification of transformant cells, i.e., cells which were competent to take up exogenous DNA. Generally, identification is by survival of transformants in culture medium that is toxic or from which the cells cannot obtain critical nutrition without having taken up the marker protein. In selecting a preferred host mammalian cell for transfection by vectors which comprise DNA sequences encoding both SNAP and DHFR, it is appropriate to select the host according to the type of DHFR protein employed. If wild type DHFR protein is employed, it is preferable to select a host cell which is deficient in DHFR thus permitting the use of the DHFR coding sequence as a marker for successful transfection in selective medium which lacks hypoxanthine, glycine, and thymidine, critical nutrients that are not available without DHFR. An appropriate host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 72, 4216 (1980) . This method is further described in U.S. Pat. No. 4,399,216.

Note that if DNA encoding DHFR protein with low binding affinity for methotrexate (MTX) is used as the controlling sequence, it is not necessary to use DHFR resistant cells. Because the mutant DHFR is resistant to MTX, MTX containing media can be used as a means of selection provided that the host cells are themselves MTX sensitive. Most eukaryotic cells which are capable of absorbing MTX appear to be methotrexate sensitive. One such useful cell line is a CHO line, CII0-K1 (ATCC No. CCL 61) .

Other methods suitable for adaptation to the synthesis of SNAP in recombinant vertebrate cell culture include those described in M-J. Gething et al., Nature 293 , 620 (1981) ; N. Mantei et al., Nature 281, 40; A. Levinson et al., EPO Application Nos. 117,060A and 117,058A.

SNAP synthesized in recombinant culture is characterized by the presence of non-human cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover SNAP from the culture. These components ordinarily will be of yeast, procaryotic or non-human higher eukaryotic origin and and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of SNAP free of homologous proteins. Homologous proteins are those which are normally associated with the SNAP as it is found in nature in its species of origin, e.g. in cells, cell exudates or body fluids. For example, a homologous protein for human SNAP is human serum albumin. Heterologous proteins are the converse, i.e. they are not naturally associated or found in combination with the SNAP in question. 4_j_. Immunocontraceptive applications.

Because SNAP is autoantigenic, it can be used as an immunocontraceptive agent. Thus, the present invention provides an immunocontraceptive method comprising administering an animal subject an immunogen selected from the group consisting of SNAP, fragments thereof, and derivatives thereof in an amount effective to reduce the fertility of that subject. The fragments and derivatives are preferably selected to bind antibodies which bind to SNAP, and also preferably bind antibodies which bind to sperm (particularly human sperm) . Partial reductions in fertility (i.e., effects which are reflected as a reduction in fertility in a population of subjects) are intended as within the scope of the present invention.

Any animal may be treated by the immunocontraceptive method of the present invention, including both birds and mammals. Exemplary mammals include mice, rabbits, dogs, cats, cows, pigs, sheep, horses, and humans. Mammalian subjects are preferred. The subject may be male or female, but male subjects are preferred. The immunogen may be administered to the subject by any suitable means. Exemplary are by intramuscular injection, by subcutaneous injection, by intravenous injection, by intraperitoneal injection, and by nasal spray. The amount of immunogen administered will depend upon factors such as route of administration, species, and the use of booster administrations. In general, a dosage of about .1 to about 100 μg per pound subject body weight may be used, more particularly about 1 μg per pound.

The immunocontraceptive method of the present invention contemplating both human and veterinary treatments, the immunogen may be prepared as both human and veterinary vaccine formulations. Vaccine formulations of the present invention comprise the immunogen in a pharmaceutically acceptable carrier. The immunogen is included in the carrier in an amount effective to reduce the fertility of the subject being treated, with the precise amount of the immunogen included depending upon the route of administration. Pharmaceutically acceptable carriers are preferably liquid, particularly aqueous, carriers, such as sodium phosphate buffered saline. The vaccine formulation may be stored in a sterile glass container sealed with a rubber stopper through which liquids may be injected and formulations withdrawn by syringe.

Vaccine formulations of the present invention may optionally contain one or more adjuvants. Any suitable adjuvant can be used, exemplary being aluminum hydroxide, aluminuum phosphate, plant and animal oils, and the like, with the amount of adjuvant depending on the nature of the particular adjuvant employed. In addition, the vaccine formulations may also contain one or more stabilizer, exemplary being carbohydrates such as sorbitol, anitol, starch, sucrose, dexrin, and glucose, proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.

5. Diagnostic applications.

The diagnostic method of the present invention provides a method of diagnosing autoimmune infertility in both male and female subjects. The term "autoimmune" is here used in a generic sense, as the immunity in female subjects is to exogenous sperm. Currently, we find such diagnostic assays to provide a superior prediction of infertility in male subjects.

Any conventional procedure for detecting serum antibodies can be employed in practicing the diagnostic assay of the present invention, including agglutination and precipitation reactions, radioimmunoassays, enzyme immunoassays (e.g., U.S. Pat. No. 3,654,090) such as Enzyme- Linked Immunoεorbent Assays (ELISA) , heterogeneous fluorescent immunoassays (e.g., U.S. Pat. Nos. 4,201,763; 4,171,311; and 3,992,631) , and homogeneous (separation-free) immunoassays. See generally Basic and Clinical Immunology, 364 (J. Fudenberg et al. eds. 3d Ed. 1980) ELISA is preferred.

In a preferred embodiment, serum from a human to be diagnosed is contacted with an antigen as described above so that antibodies in the serum react in solution with the antigen. The order of addition of these components is not critical. While the antigen is preferably bound to a solid support, if a homogeneous (separation-free) immunoassay is utilized to detect the antibodies, a solid support would not be required. Serum may be obtained from a person generally by pricking a finger and obtaining whole blood (of which serum is a constituent) . However, the blood may be processed to obtain only the serum or plasma portion of the whole blood before contacting the serum with the bound antigen. Any method for obtaining serum or plasma from a patient may be utilized as long as the antibodies contained therein retain their ability to bind the antigen.

The antigen employed in the diagnostic assay is, as noted above, selected from the group consisting of (a) SNAP, (b) fragments of SNAP, and (c) derivatives of either SNAP or SNAP fragments which bind selection antibodies which bind to SNAP (and preferably also bind antibodies which bind to sperm) . The antigen is preferably substantially free of extraneous proteins. The antigen may be bound to solid supports by known techniques. For example, a bi-functional organic molecule may be used to attach the antigen to a solid support. The solid can be made of materials such as plastic (e.g., the bottom surface of a well in a microtiter plate) , fiberglass, cellulose acetate and nitrocellulose (e.g., discs) . After being attached or adhered to the solid support, the antigen can be cross-linked if desired.

The step of contacting the solid support with a detectable antibody is carried out so that the detectable antibody is allowed to interact with the antigen bound to the solid support. The detectable antibody is one which is capable of binding to a human antibody from the serum of the patient which has bound the purified antigen, where the detectable antibody is capable of being detected. More particularly, the detectable antibody can be an anti-human immunoglobulin which is conjugated to a group such as an enzyme which is detectable in the presence of a substrate. Enzyme-conjugated goat or rabbit anti-human antibodies which have been affinity purified are preferred. In general, the detectable group which is conjugated to the detectable antibody may be any enzyme or other detectable species which has been developed for immunoassays. For example, enzymes, fluorescent groups, radioactive groups and others could be used. The enzyme peroxidase is particularly preferred. When peroxidase is the detectable group conjugated to the detectable antibody, a substrate such as 3, 3' , 5, 5'-tetra- methylbenzidine or o-phenylenediamine may be used as the substrate for detection of the detectable antibody.

The step of detecting the detectable antibody that has reacted with the human antibodies involves treating or manipulating the detectable group which is conjugated to the detectable antibody so as to determine its presence. For example, if an enzyme such as peroxidase is conjugated to the antibody, the detecting step would involve adding a peroxidase substrate to the bound antibody and observing a color change as peroxidase catalyzes conversion of the substrate to a colored species. In the case of other enzymes, such as alkaline phosphatase and /3-D-galactosidase, other substrates may be used. The substrate to be used should be chosen such that after the enzyme catalyzes a chemical conversion of the substrate to a product, a change which is observable to a person employing this test should result. Substrates such as 3 ,3 ' , 5, 5'-tetramethylbenzidine, p-nitrophenyl phosphate or 3, 3 '-diamino-benzidine may be used as substrates. Other detectable groups may also be conjugated to the antibody.

A kit containing the required components for carrying out a diagnostic test based on detection of serum antibodies can be assembled. The kit comprises a package containing purified antigen coated in or on a solid support such as the bottom of a microtiter plate well or a nitrocellulose or cellulose acetate disc, and a container of a detectable antibody conjugate which is capable of binding antibody from the serum of a patient which is bound to the antigen. An ELISA test is most preferred for the kit since it lends itself to a readily detectable positive or negative diagnosis. Thus, the kit should also house a container of a substrate which is reactive with an enzyme which is conjugated to the detectable antibody, the substrate being readily detectable after reaction with the enzyme.

6. Examples.

The examples provided below are illustrative of the invention, and are not to be taken as limiting thereof.

Materials. New Zealand White rabbits and Balb/c mice used for immunization and mRNA isolations were obtained through the Department of Laboratory Animal Medicine at the University of North Carolina at Chapel Hill. Esherichia coli (E. coli) strains Y1088, Y1090, LE392, DH5α and JM109 were obtained from the Laboratories for Reproductive Biology Recombinant DNA Core Facility at UNC-Chapel Hill; an EMBL-3 rabbit genomic library and E. coli Y1089 were purchased from Clontech laboratories (Palo Alto, CA) ; and E. coli JM101 was obtained from the ATCC (Rockville, MD) . Cultures were maintained according to T. Maniatis et al. , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982) . [³²P] and [³⁵S] labelled nucleotides and deoxynucleotides were obtained from Λmersham (Arlington Heights, IL) and ICN (Irvine, CA) . Kodak XAR-5 x-ray film (Rochester, NY) and Cronex Lightning-Plus screens (DuPont, Wilmington, DE) were used for autoradiography. All chemicals were of reagent or molecular biology grade unless otherwise indicated.

Electrophoresis and blotting. DNA samples were separated by submerged agarose gel electrophoresis under standard nondenaturing conditions using a Tris-borate-EDTA buffer system and stained with ethidium bromide (T. Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982) . Hind III digested lambda DNA and Hae III digests of PhiX174 DNA were used as markers (Bethesda Research Laboratories) . Southern blotting to nitrocellulose (Schleicher and Schuell) and hybridization to [³²P] labelled DNA were performed according to G. Wahl et al . , Proc. Natl . Acad. Sci. 76, 3683 (1979) . RNΛ samples were electrophoresed through agarose gels containing formaldehyde, transferred to nitrocellulose and hybridized to [^3ZP] labelled DNA as described (S. Thomas, Proc. Natl. Acad. Sci. USA 77, 5201 (1980)) . [³²P] labelled DNA used as probe was prepared by nick translation (P. Rigby et al., J. Mol. Biol. 113, 237 (1977)) using reagents obtained from Bethesda Research Laboratories. Exposures were performed at -70°C. SDS-PΛGE and immunodetection of proteins were carried out according to the procedures of U. Laemmli, Nat. New Biol. 227. 680 (1970) and II. Towbin et al., Proc. Natl. Acad. Sci. USA. 76, 4350 (1979) and as described previously (M. O'Rand and J. Porter, Biol. Reprod. 27, 713 (1982) ; and J. Welch and M. O'Rand, Develop. Biol. 109, 411 (1985)) .

EXAMPLE 1

Production of Rabbit Anti-Spermatozoa Autoantiserum

And Anti-RSA Alloantiserum

The production of rabbit anti-spermatozoa autoantiserum and anti-RSA alloantiserum has been described previously (M. O'Rand and J. Porter, J. Immunol■ 122, 1248

(1979) ; and M. O'Rand and J. Porter, Biol. Reprod. 27. 713 (1982)) . Alloantiserum depleted of anti-RSA activity was prepared by repeated incubations with purified RSA bound to nitrocellulose membrane (Schleicher and Schuell, Keene Nil) . Absorptions were continued until no anti-RSA activity remained as judged by ELISA (M. O'Rand et al. , Develop. Biol. 129, 231

(1988) using peroxidase conjugated goat anti-rabbit IgG

(Cooper Biomedical, Malvern, PA) .

EXAMPLE 2 RNA And DNA Isolation Total RNA and mRNA were isolated by the procedure of J. Chirgwih et. al., Bioche . 18, 5294 (1979), using either fresh tissue or material quick frozen at -195^*C and stored under liquid nitrogen. Frozen tissue was reduced to a powder with a mortar and pestle before use. Lambda DNA was purified by the method of D. Kaslow, Nucl. Acids Res. 14, 6767 (1986),- digested with various restriction enzymes (Bethesda Research Laboratories, Gaithersburg, MD and Boehringer Mannheim, Indianapolis, IN) , and DNA fragments separated by submerged agarose gel electrophoresis. DNA fragments were recovered by binding to and elution from DEΛE paper (Schleicher and Schuell) as per instructions.

EXAMPLE 3 Construction of cDNA Library and Selection of Partial SNAP cDNA Clones Our original goal was to isolate cDNA sequences coding for Rabbit sperm membrane autoantigen (RSA) . mRNA from isolated rabbit seminiferous tubules was used to synthesize blunt ended, double stranded cDNA by the ribonuclease H procedure (U. Gubler and B. Hoffman, Gene 25, 263 (1983)) . This cDNA was cloned into the bacteriophage lambda gtll expression vector to produce a library of about 500,000 recombinants with an average size of 1200 bp. See R. Young and R. Davis, Proc. Natl. Acad. Sci. USA 80, 1194 (1983) ; and R. Young and R. Davis, Science 222, 778 (1983) . Positive cDNA clones were isolated by screening for RSA epitopes with the IgG fraction of rabbit anti-RSA alloantibody. Initial positive clones were rescreened with anti-RSA IgG absorbed with purified RSA to confirm the presence of depletable RSA epitopes in the encoded fusion protein. Only those clones whose antibody binding activity was abolished by RSA absorption were selected for further study.

An initial screening of the rabbit seminiferous tubule library was undertaken to isolate a cDNA encoding RSA. The library was therefore screened with rabbit anti-RSA alloantibodies and three positive clones were isolated. These three clones were rescreened with anti-RSA depleted alloantiserum and only one clone, designated R1.2, failed to react with the absorbed antibody. Rl.2 was subsequently determined to contain a cDNA insert of 1.5 kbp and by Southern blot analysis demonstrated no cross-hybridization with the remaining two clones, a feature further demonstrated by direct comparison of the three DNA sequences. Rescreening of these clones with an oligonucleotide derived from an RSA peptide sequence:

( (5' ) CCG CCG CCG TGG .AC GGG SG SG CCG CCG ACC ACC TSG (3')) labelled with [³²P] and additional anti-RSA antibodies showed that these clones did not encode a genuine RSA sequence. Further hybridization screening of both rabbit seminiferous tubule cDNA and genomic libraries with R1.2 insert DNA as a probe was employed to find additional clones and determine the complete nucleic acid sequence of SNAP (see below) .

EXAMPLE 4

Northern Blotting of RNA from Various Sources

With Partial SNAP cDNA Clone

Northern blots of total RNA from rabbit testis, probed with [³²P] labelled R1.2 cDNA, showed strong hybridization with a 2.5 kb mRNA species. It was evident from a size comparison of the R1.2 insert and its detected mRNA that R1.2 represented a partial clone of the 2.5 kb mRNA. This mRNA was not detected in RNA isolated from male rabbit liver, brain, spleen, heart, and kidney, as well as female rabbit liver and ovary. Northern blot analysis of mouse RNA from ovary, uterus/oviduct, heart, skeletal muscle, skin, stomach, kidney, thymus, seminal vesicle, epididymis, spleen, liver, preputial gland and pancreas indicated the absence of SNAP mRNA in these tissues. Hybridization of the R1.2 cDNA probe with blots of mouse and rat testis RNA revealed similar mRNA's.

EXAMPLE 5

Isolation of Genomic SNAP Clones by Hybridization Screening with Partial SNAP cDNA Sequence Because only partial clones of SNAP were obtained from the cDNA library, genomic clones encoding SNAP sequences were isolated by hybridization screening (W. Benton and R. Davis, Science 196, 180 (1977)) of a rabbit genomic DNA library with a partial SNAP cDNA. The presence of 5' genomic sequence was confirmed by restriction mapping and Southern blotting using an oligonucleotide probe complementary to nucleotides (nt) 718-757 of the mRNA. A 3.85 kb Eco Rl fragment and a 4.2 kb Pvu II fragment both containing the 5' genomic regions were subcloned into M13mpl9 for later sequencing.

EXAMPLE 6 DNA Sequencing and Analysis

All sequencing was done by the dideoxynucleotide chain termination method (F. Sanger et al., Proc. Natl. Acad. Sci. USA 74. 5463 (1977)) using the Klenow fragment of DNA polymerase I (BRL) or a modified T7 DNA polymerase or a Taq DNA polymerase (United States Biochemical Corp., Cleveland, OH) . DNA was subcloned into either M13mpl8 or M13mpl9 vector DNA (BRL, Boehringer Mannheim, Indianapolis, IN; C. Yanisch- Perron et al., Gene 33 , 103 (1985)). Clones containing unidirectional deletions were made with T4 polymerase (International Biotechnologies, New Haven, CT; R. Dale et al., Plas id 13 , 31 (1985)) and used for sequencing both orientations of the cDNA clone and the antisense strand of the genomic Pvu II fragment. The genomic sequence was confirmed by sequencing the genomic Eco RI fragment using synthetic oligonucleotides as primers. A synthetic oligonucleotide (nt 729-745) was made to the 5'end of the 1456 C-terminal clone (R1.2) and used as a primer on the antisense strand of a genomic clone (RG1) to extend 338 bp upstream. A second oligonucleotide (nt 381-397) was synthesized and used to extend an additional 400 bp upstream.

The beginning of the cDNA was determined by primer extension (R. Kingston, Primer Extension. In Current Protocols in Molecular Biology. F. Ausubel et al. editors. John Wiley & Sons, New York. 4.8.1-4.8.3., 1987) using an end labeled synthetic oligonucleotide (nt 11-50) . The primer was extended using cold deoxynucleotides and the extension product analyzed on a sequencing gel using M13Mpl8 as a size standard. Gel electrophoresis and autoradiography were performed according to instructions supplied (Bethesda Research Laboratories; Bio-Rad, Richmond, CA) .

Sequence analyses and comparisons were performed using software supplied by Intelligenetics (Mountain View, CA) . Hydrophilicity analyses (T. Hopp and K. Woods, Proc. Natl. Acad. Sci. USA 78, 3824 (1981)), secondary structure predictions (P. Chou and G. Fasman, Adv. Enzymol. 47, 45 (1978) ; J. Gamier et al., J. Mol. Biol. 120, 97 (1978) ; and M. Schiffer and A. Edmunsen, Biophys. J. 7_, 121 (1967) , and sequence comparisons (D. Lipman and W. Pearson, Science 227 , 1435 (1985) ; J. Pustell and F. Kafatos, Nucl. Acids Res. 10, 4765 (1982) ; and J. Pustell and F. Kafatos, Nucl. Acids Res. 12 , 643 (1984), used the methods indicated.

Figure 1 shows the complete sequence of the SNAP DNA sequence based upon cDNA and genomic sequences. The genomic sequence also indicated the presence of an intron between bases 110 and 111. The intron was initially identified by the loss of open reading frame and the presence of flanking splice sites (S. Mount, Nucl. Acids Res. 10, 459 (1982)). The location was further confirmed by comparison with a human SNAP cDNA encoding the entire protein (see Fig. 2) . The first potential initiation methionine meets consensus sequence requirements (M. Kozak, Cell 44, 283 (1986)) with the ΛTG initiation codon being preceeded by an A at -3 upstream. The reading frame was terminated at the 3 ' end by an ochre stop codon (TAA) followed by 104 nucleotides of untranslated sequence. This untranslated region contained a poly (A) addition signal (AATAAA) which was located 10 bases from the beginning of a poly (A) tail. A second potential poly (A) addition sequence begins at base 1180, but has not been seen to function in the testis. The SNAP DNA coding sequence of Figure 1 encodes an acidic protein of 680 AA with a calculated molecular weight of 73,533 daltons and pl=4.06. Computer analysis predicted that the putative SNAP protein sequence has a large alpha-helical content with 69% of the molecule displaying a strong propensity to form helices. A repeating heptad pattern, (a-b-c-d-e-f-g)n, where amino acids a and d are usually hydrophobic, was also noted. The putative SNAP protein appeared generally hydrophilic and contained 25% charged residues. One region, spanning amino acids 354-404, was very hydrophilic, containing 49% acidic amino acids. A second, smaller sequence (AA 16-26) was even more acidic, containing 73% glutamic acid. A potential nuclear translocation signal was located between amino acids 609-615. This basic peptide sequence (VRKKRKPE) displayed significant similarity to the putative nuclear localization signal of the SV40 large T antigen (PKKKRKVE; A. Smith et al., Proc. R. Soc. Lond. 226, 43 (1985) .

COMPARATIVE EXAMPLE A

Sequence Similarity with Xenopu3 N1/N2

A comparison of the deduced SNAP amino acid sequence with other protein sequences in the Protein Identification

Resource (NBRF) and the SWISS-PROT database (A. Bariroch, SWISS-PROT Release 6.0 (University of Geneva, Geneva,

Switzerland) (1988)) yielded mostly low percentage matches with proteins of high glutamic acid content. However, a significant similarity was found between SNAP and the Xenopus laevis histone-binding protein, N1/N2. Most of the similarity was in the last 275 amino acid residues (AA) of SNAP ( AA 401-680) which exhibited a 60% identity with the N1/N2 polypeptide. The SNAP and N1/N2 sequences prior to residue 401 exhibited very little sequence identity (15%) even when large gaps are introduced to maximize sequence matches. SNAP residues 601-645 were 80% identical with N1/N2 residues 531-537. This region has been identified as containing one and possibly two nuclear translocation signals (J. Kleinschmidt and A. Seiter, EMBO J. 7. 1605 (1988)).

Two highly acidic, histone-binding regions have been identified in N1/N2 as residues 108-119 and 296-326 (Kleinschmidt and Seiter, supra) . The larger histone binding region is located in the same region as one highly acidic SNAP sequence (ΛA 354-404) . While these areas could represent functionally similar regions (33% identity at glutamic acid residues) , we have no evidence for histone-binding by SNAP. The second, smaller N1/N2 histone-binding region (ΛA 108-119) does not appear to be conserved in SNAP, although SNAP does contain a second glutamic acid rich sequence near the amino terminus (AA 16-26) .

The SNAP and N1/N2 nucleic acid residues were approximately 80% similar within the region encoding SNAP amino acid residues 401-680, immediately downstream from the internal poly (A) addition signal. No significant matches were found between N1/N2 and the nucleotide sequence coding for amino acid residues 1-400 of SNAP. The SNAP and N1/N2 3' untranslated regions also showed some similarity (60%) beginning approximately 50 bases upstream from the poly (A) addition signal.

EXAMPLE 7 Production of Recombinant SNAP Fragment Fusion Protein with 3-Galactosidase

The cDNA clone R1.2 (1446 bp; nt 703-2148) was used to produce a fusion protein (fpR1.2) containing the C-terminal 446 amino acids of SNAP. Bacteriophage lambda GT11 was the vector and rl.2 was inserted in the vector in the lacZ Eco Rl restriction site. Rl.2//3-galactosidase fusion protein (fpR1.2) and /.-galactosidase (/3-gal) production was induced in lysogenic Y1089 strain E. coli in accordance with known techniques. See T. Huynh et. al., .in 1 DNA Cloning, 49-78 (D. Glover ed. 1985) .

EXAMPLE 8 Production of SNAP Antisera

A bicinchoninic acid protein assay (A. Smith et al., Proc. R. Soc. Lond. 226, 43 (1985) ; Pierce Chemical Company, Rockford, IL) was used to determine the amount of total protein present. Recombinant fusion proteins were produced as described in Example 7 above and isolated by SDS polyacrylamide gel electrophoresis. The fpR1.2 fusion protein band was cut from a fixed, stained 7% SDS polyacrylamide gel, washed in several changes of PBS, and pulverized by repeated passage through an 18 gauge needle. Female mice were immunized with 150 μl (lOμg of protein) of gel mixed 1:1 with complete Freund's adjuvant and boosted at 4 and 6 weeks. Control mice received blank gel with 10 ug of purified E. coli /3-gal (Sigma, St. Louis, MO) and the appropriate adjuvant. All antisera were absorbed with an E. coli lysate containing 3-gal in order to minimize antibody binding to /3-gal or E. coli proteins. Antisera were also absorbed with human cytokeratin to eliminate any potential binding due to minor contamination by these proteins (S. Shapiro, J. Immunol. Meth. 102, 143 (1987). Antiserum raised in mice against the R1.2//3- galactosidase fusion protein (anti-fpR1.2) was tested in ELISA for binding to E. coli lysate containing fpR1.2 and exhibited a high titer (>1:25,000) against fpR1.2, while anti-/3- galactosidase (anti-?-gal) exhibited appreciable binding at only the lowest dilutions (<1:400) .

Western blots of E. coli lysates containing either j0-gal or fpR1.2 showed the expected upward shift of the }-gal band after the insertion of the R1.2 cDNA. Although the predicted M_r of the fpRl.2 was 163 kD, including 114 kD of /3-gal sequence, on SDS-PAGE it migrated with M_r=185 kD. The predicted size contribution of the R1.2 insert was 49 kD, but the observed increase was 71 kD. The aberrant migration is probably due to a high percentage of acidic residues present in the protein. E. Kaufmann et al., FEBS Letters 170, 81 (1984) . Western blots of E. coli lysate were also probed with rabbit anti-rabbit sperm autoantiserum. Staining of fpR1.2 with anti-sperm autoantisera indicated the presence of autoantigenic epitopes in the R1.2 polypeptide. The adjuvant control serum showed no staining of the fpR1.2 band. Some E. coli proteins were stained with both anti-sperm and adjuvant control sera, but no staining of the /3-gal band was seen using either serum.

EXAMPLE 9 Presence of SNAP in Rabbit Testis Western blot analysis of SNAP in rabbit testis using the Rl.2 fusion protein antisera as a probe showed a major band at M_r=130 kD (pl30) on western blots. Three closely spaced doublets ranging from 124-109 kD were detected immediately below the major pl30 band despite the presence of protease inhibitors. Additional minor bands at M_r=73 (p73) , 69 kD (p69) and 64 kD (p64) were consistently detected, but they always labelled less intensely than the pl30 protein. In order to determine if SNAP was present in testis membrane fractions, western blots of membrane protein were also probed with anti-fpR1.2. Immunostained bands identical to the pl30 and associated doublets from whole testis were detected, indicating the presence of pl30 in testis membrane preparations. Minor proteins at 94, 81, 69, and 60 kD were also labelled, but much more faintly than the pl30 band. Proteins similar to pl30, p73, p69, and p64 were also detected in rat and mouse whole testis.

Immunostaining of ejaculated rabbit sperm protein labelled two sets of triplet bands, one group (I) at Mr=73, 69, 60, and a second group (II) at Mr=51, 49, and 46 kD (lanes 8-10), but no pl30 band could be detected. The 51, 49, and 46 kD bands were not previously detected among the testis proteins, while the 73 and 60 kD bands were more prominent in the sperm protein samples. A very small amount of the 130 kD protein was occasionally seen in rabbit epididymal spermatozoa as detected by immunoblotting. Λnti-β-gal control blots demonstrated no specific binding to any protein sample. The observed length of the SNAP mRNA transcript is

2.5 kb which encodes a polypeptide of molecular weight 73,533. However, the major protein observed in the testis by immunoblotting has an apparent molecular weight of 130,000 (pl30) . Consequently, it is likely that SNAP'S migration on SDS gels is anomalous. Numerous nuclear proteins display similar anomalous migration on SDS gels. Nucleolin, for example, migrates at a M_r 30% higher than its true molecular weight (B. Lapeyre et al., Proc. Nat. Acad. Sci. USA, 84 , 1472 (1987)) ; nucleoplasmin migrates 50% higher (C. Dingwall et al., EMBO J. 6_., 69 (1987)), and the greatest deviation is found in the Xenopus nuclear protein, N1/N2, with a M_r 60-70% higher than its actual molecular weight (J. Kleinschmidt et al., Embo J. 5_, 3547 (1986) . Most of the anomalous migration of these proteins has been attributed to the high percentage of acidic residues contained within the protein sequence. Since the amino acid sequence of SNAP contains 25% acidic residues and displays a substantial sequence similarity with N1/N2, it appears likely that the migration of SNAP on SDS-PAGE does not reflect its true molecular weight.

EXAMPLE 10

Isolation of DNA Sequence Coding for Human SNAP

The Human cDNA SNAP clone was obtained by following the same procedures set forth in Example 3, except that a human testis cDNA library (obtained from Clonetech Laboratories, Inc., Palo Alto, CA) was substituted for the rabbit cDNA library. Unlike our experience with rabbit, the complete human SNAP coding sequence was found in the cDNA library. The complete human SNAP cDNA clone is shown in Figures 2A to 2B.

EXAMPLE 11 ELISA Procedures

ELISA was carried out in accordance with known procedures. See, e.g.. M. O'Rand et al., Dev. Biol. 129, 231 (1988) . In brief, 1 microgram of the SNAP fusion protein prepared in Example 7 above was fixed to the bottom surface of wells in 96 well microtiter plates by cross linking with Bouin's fixative in accordance with known techniques. See, e.g.. W. Noteboom et al., J. Immunol. Methods 75. 141 (1984). Peroxidase-labelled goat anti-rabbit and anti-human IgG was used as the detectable antibody and 3,3' ,5,5'-Tetramethyl- benzidine (TMB) was used as the substrate. The TMB reaction product was read as a yellow product after the addition of 1 Normal HC1.

EXAMPLE 12 Reactivity of Antisera from Infertile Men with SNAP Because SNAP is an autoantigenic protein, antisera from a vasectomized male was tested for reactivity to SNAP. Figure 3 shows the results of an ELISA on a serially diluted serum sample from a vasectomized male. O.D. at 492 is shown on the vertical axis; 1/serum dilutions is shown on the horizontal axis. For each dilution human sperm lysate is shown on the left; fusion protein is shown on the right. ELISA was carried out in accordance with the procedures given in Example 11. In the vasectomized sera antibodies to SNAP can be detected. This data indicates SNAP is useful as an immunocontraceptive and diagnostic agent.

The foregoing examples are illustrative of the present invention, and are not to be taken as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. An essentially pure DNA sequence which codes for sperm nuclear autoantigenic protein (SNAP) or a fragment thereof.

2. An essentially pure DNA sequence according to claim 1 which codes for rabbit SNAP or a fragment thereof.

3. An essentially pure DNA sequence according to claim 1 comprising the DNA sequence shown in Figure 1.

4. An essentially pure DNA sequence according to claim 1 which codes for human SNAP or a fragment thereof.

5. An essentially pure DNA sequence according to claim 1 comprising the DNA sequence shown in Figure 2.

6. A gene transfer vector containing the DNA sequence of claim 1.

7. A gene transfer vector according to claim 6, which vector comprises a bacteriophage vector.

8. A microbial host transformed by the gene transfer vector of claim 7 containing the DNA sequence coding for SNAP.

9. A microbial host according to claim 8, which host is Escherichia coli.

10. A process for the preparation of SNAP, comprising culturing the transformed microbial host of claim 8 under conditions suitable for the expression of SNAP and recovering SNAP from the cultured host.

11. SNAP produced by the process of claim 10.

12. Essentially pure Sperm Nuclear Autoantigenic Protein (SNAP) .

13. An immunocontraceptive method comprising administering an animal subject an immunogen selected from the group consisting of Sperm Nuclear Autoantigenic Protein (SNAP) , SNAP fragments, and derivatives thereof which bind selection antibodies which bind to SNAP, in an amount effective to reduce the fertility of said subject.

14. An immunocontraceptive method according to claim 13, wherein said subject is a mammalian subject.

15. An immunocontraceptive method according to claim 13, wherein said subject is a male subject.

16. An immunocontraceptive vaccine formulation comprising an immunogen selected from the group consisting of Sperm Nuclear Autoantigenic Protein (SNAP) , SNAP fragments, and derivatives thereof which bind selection antibodies which bind to SNAP, in an amount effective to reduce the fertility of an animal subject in combination with a pharmaceutically acceptable carrier.

17. An immunocontraceptive vaccine formulation as claimed in claim 16, further comprising an adjuvant.

18. An immunocontraceptive vaccine formulation as claimed in claim 16, further comprising a stabilizer.

19. An immunocontraceptive vaccine formulation as claimed in claim 16, wherein said derivatives are selected from the group consisting of SNAP and SNAP fragments having (a) one or more amino acid residues changed, (b) one or more amino acid residues deleted, or (c) one or more amino acid residues changed and one or more amino acid residues deleted.

20. A method of diagnosing autoimmune infertility in a human subject comprising:

(a) contacting an immune sera sample from the subject with an antigen, the antigen selected from the group consisting of Sperm Nuclear Autoantigenic Protein (SNAP) , SNAP fragments, and derivatives thereof which bind selection antibodies which bind to SNAP, and then

(b) detecting the presence of antibodies from the immune sera sample bound to the antigen, the presence of antibodies bound to the antigen suggesting the subject is afflicted with autoimmune infertility.

21. A diagnostic method according to claim 20, wherein said subject is a male subject.

22. A diagnostic method according to claim 20, wherein said derivatives are selected from the group consisting of SNAP and SNAP fragments having (a) one or more amino acid residues changed, (b) one or more amino acid residues deleted, or (c) one or more amino acid residues changed and one or more amino acid residues deleted.

23. A test kit useful for diagnosing autoimmune infertility in a human subject by detecting serum antibodies in a serum sample from that subject, the kit comprising a housing which accomodates: (a) a solid support having an antigen bound thereto, the antigen selected from the group consisting of Sperm Nuclear Autoantigenic Protein (SNAP) , SNAP fragments, and derivatives thereof which bind selection antibodies which bind to SNAP; and (b) a container containing detectable antibodies which bind to said serum antibodies.

24. A kit according to claim 23, wherein said solid support is a well having said antigen contained therein.

25. A kit according to claim 24, wherein said detectable antibodies comprise antibodies bound to an enzyme.

SUBSTITUTE SHEET