KR20000075624A - Method for the Production of Vaccines Against Cell Surface Proteins - Google Patents

Abstract

The present invention relates to a method for identifying a repertoire of proteins exposed to the surface of a virus, bacterium or cell and a method for preparing a vaccine therefor. Identification methods include directionally labeling proteins on the membrane surface, isolating the labeled membrane surface proteins by two-dimensional gel electrophoresis, and sequencing the isolated membrane surface proteins. Also disclosed are methods of making vaccines against viruses, bacteria or cells, methods of detecting infertility, methods of inducing contraception, and vaccines and contraceptives prepared by these methods.

Description

Methods for the production of vaccines against cell surface proteins {Method for the Production of Vaccines Against Cell Surface Proteins}

Government assistance

The study was funded in part by grants NIH HD 29099 and P30-28934 from the National Institutes of Health (NIH). The US government has certain rights in the invention.

Background of the Invention

Living organisms interact with their environment through their surfaces. The immune system elicits antibody mediated and T cell mediated immune responses to epitopes present on cell and viral surfaces. Since the immune response is elicited against the cell / virus surface, a method that enables the identification, isolation and genetic manipulation of surface molecules of living organisms is a means of developing vaccines.

Mammalian sperm are highly differentiated cells. Known as the cycle of spermatogenic epithelium, it is the product of a complex series of changes that are spatially and temporarily reorganized into a set of cell populations (1). During spermatogenesis, undifferentiated rounded garden cells are converted into highly asymmetrical and highly motile sperm cells through the spermatogonia and the spermatogonia, and in humans 12 steps are known in the differentiation of sperm cells alone (2). As a result of this numerous differentiation, sperm become unique in many ways. Nuclei chromosomes are highly condensed and inactivated in RNA synthesis, resulting in unique cellular components such as the tip of the sperm head and the outer dense fibers and fibrous ribs of the tail. Although not immediately visible in the microscopic view of sperm, even the sperm cell membrane undergoes significant differentiation during spermatogenesis. Different membrane domains are formed on the sperm surface, such as those found in immunocytochemical studies on differences in the distribution of sperm membrane components (5) and those observed in frozen crushed specimens (4). Moreover, the sperm surface does not stop after leaving the spermatogenic epithelium, and its components undergo transformation and redistribution as a result of further maturation in the epididymis, followed by fertility acquisition and peak body reactions in the female reproductive tract.

When sperm moves from the testicle to the fallopian tube, it is through his surface that the male and female tubes interact with the surrounding environment. The most important sperm cell membrane (protoplasm) is in contact with the egg coat, and the membrane that surrounds the equatorial compartment of the tip is believed to be the initial site of fusion with the egg cell membrane.

Recently, Chong Xu et al. (26) found that 64 plasma membrane silver stained proteins can be extracted using a detergent from a human sperm membrane vehicle separated by nitrogen cavitation, differential centrifugation, and IEF / PAGE electrophoresis. Reported. However, in the analysis of sperm surface proteins, nitrogen cavitation techniques have the drawback of having to accumulate samples over several weeks to obtain a sufficient amount of starting material. In addition, uncertainty remains whether cavitation methods yield "membrane" proteins derived only from cell membranes (20).

In the past, two-dimensional electrophoresis was used to study human sperm proteins (18-26), but due to lack of standardization of procedures, it was difficult to adjust protein patterns between these previous reports. Comprehensive classification of human sperm surface proteins has limited data available in one or more of: (1) resolution, (2) reproducibility, (3) pH range, (4) specificity of surface labeling technology, and (5) cell membrane purification methods. Because I was in trouble.

Given the above deficiencies attached in accordance with prior art methods of analyzing cell surface proteins, broadly the standardized methods of analysis, identification, characterization, isolation and classification of cell surface proteins, more specifically sperm surface proteins, What the industry needs is clear.

Summary of the Invention

Therefore, the object of the present invention

(a) vectorially labeling the protein on the membrane surface,

(b) isolating the labeled membrane surface protein by two-dimensional gel electrophoresis, and

(c) to provide a novel method for analyzing membrane surface proteins comprising analyzing the sequence of isolated membrane surface proteins.

Another object of the present invention

(a) directionally labeling the protein on the membrane surface,

(b) isolating the labeled membrane surface protein by two-dimensional gel electrophoresis,

(c) sequencing the isolated membrane surface protein,

(d) cloning the DNA encoding the membrane surface protein, and

(e) providing a method for preparing a vaccine against the membrane surface protein, comprising the step of recombinantly preparing the membrane surface protein using the DNA isolated in step (d).

Another object of the present invention

(a) labeling the membrane surface protein obtained by the above method,

(b) reacting the labeled membrane surface protein with serum obtained from a patient to be diagnosed for infertility, and

(c) detecting the formation of a complex between the antibody present in the serum and the labeled membrane surface protein.

Another object of the present invention is to provide a vaccine prepared by the above method.

Another object of the present invention is to provide a diagnostic test kit comprising the cell surface protein prepared by the above method.

Another object of the invention is to induce contraception in a patient in need thereof, comprising administering sperm cell surface protein prepared by the method in an amount sufficient to interfere with fertilization of the egg in the patient. Provide a way to.

It is a further object of the present invention to provide a method of preparing a contraceptive comprising administering to a mammal a recombinant protein produced by the above method and isolating an antibody against said recombinant protein produced by said mammal.

Another object of the present invention is to provide a contraceptive prepared by the above method.

The foregoing, and other objects, advantages and features of the present invention will become apparent from now on, and the nature of the present invention will be more clearly understood by reference to the detailed description of the preferred embodiments of the present invention and the appended claims. will be.

The present invention broadly relates to a method for identifying a protein repertoire exposed to a cell surface and a method for preparing a vaccine therefor. Specifically, it relates to a method for identifying a protein repertoire present on the surface of human sperm, a protein identified by this method, and a method for preparing a contraceptive vaccine therefrom.

A more complete understanding of the present invention and the many advantages associated with the present invention will be gained when it is better understood with reference to the following detailed description, taken in conjunction with the accompanying drawings.

1 shows the results of two-dimensional gel electrophoresis analysis of sperm plasma proteins [C and D] obtained from silver-stained human sperm [A and B] solubilized in lysis buffer A and in atypical patients (see method). Proteins were separated by IEF / PAGE [A and C] and NEPHGE / PAGE [B and D], resulting in a resolution of approximately 1300 sperm protein spots. The pH gradient of the first dimension gel is shown at the top of the figure. Note that the pH range between the two first-dimensional techniques overlaps. SDS-PAGE was performed so that the lower layer of the gel became the anode. The molecular weight (x 10 ^-3 ) was labeled in the left margin. There are relatively few high molecular weight proteins in the liquefied semen plasma [C and D], while numerous protein spots of 20 kDa or less appear. The pattern of degradation of seminal plasma proteins may appear following the oblique pushes observed between the basic proteins of D (horizontal arrows). Computer comparison of sperm plasma and sperm protein patterns revealed nine co-moving proteins of similar shape (black and white arrows). White arrows in A and C clearly indicate the position of albumin that is anchored to the sperm surface.

2 shows the difference in the amount of 39.5 kDa protein in sperm obtained from three healthy young men. Three samples were treated identically by simultaneous electrophoretic separation and the like in the same buffer tank. The figure shows an enlarged area of three IEF / PAGE gels. The estimated proportion of silver stained 39.5 kDa protein (indicated by arrow) varied from 0.412 of A to 3.703 of C. Such concentration differences were also observed between surface proteins (FIG. 7).

Figure 3 is the result of studying the amount of radioactive iodine incorporation into human sperm protein over time. Four aliquots of 30 × 10 ⁶ Percol-recovered sperm from the same donor were radiolabeled using 10 iodine-beads (IODO-BEAD) in 4 ml of Dulbecco's PBS per aliquot and Na ¹²⁵ I 150 μCi. Cells were removed from the catalyst beads by pipette to stop iodine incorporation after 5, 10, 15 and 20 minutes. Sperm were pelleted by centrifugation and the amount of iodine incorporated was counted by a gamma counter and compared with the amount of iodine in the reaction buffer.

FIG. 4 shows a comparison of labeled sperm protein (A) extracted from Lysis Buffer A without pretreatment with labeled sperm protein extracted from Lysing Buffer A centrifuged (B) lysed after Percoll density centrifugation. The number of labeled proteins was small in sperm samples after the second Percoll isolation (B). The labeled protein in Figure A included the polymorphic tip protein SP-10 (horizontal arrowhead) and actin (white arrow), indicating that the cytoplasmic component was labeled (see Figure 6 for an immunoblot for SP-10). ). In contrast, the control cytoplasmic marker was not labeled in Figure B. Therefore, Percoll centrifugation after surface labeling was clearly considered essential for the analysis of surface proteins only.

5 (FIGS. 5A-5B) show Western blots reacted with monoclonal antibodies against cytoplasmic components with two-dimensional IEF PAGE of directionally iodinated human sperm protein. Sperm from one donor was radioiodized, Percoll centrifuged, and solubilized in [C and D] buffer A in non-reducing conditions [A and B] or in the presence of a reducing agent (100 mM DTT). Ampoline compositions of 28% pH 3-3.5, 20% pH 5-7, 7% pH 7-9 and 45% pH 3.5-10 were used to improve the resolution of acidic proteins. A of FIG. 5A is a magnetic radiograph of the non-reduced protein immobilized on the NC membrane. The position of the actin in the radioiodination pattern is indicated by an arrow. FIG. 5B shows Western blot of the protein of FIG. 5A A incubated with mAb prior to autoradiophotography treatment. Four isoforms of actin were resolved at the expected molecular weight of 43 kDa with a pi of 5.1 to 5.2. In B1 of FIG. 5A, the radioradiograph is at the top of the corresponding immunoblot. The wide area clearly shows that monomeric actin is not iodide. Horizontal arrows in B1 and B2 of FIG. 5A indicate the location of two proteins, 51 and 52 kDa, which immunoreactively react with actin. These proteins stained very weakly with a 1: 3000 dilution of the antibody (FIG. 5A), but were more apparent when the antibody was used at a 1: 1500 dilution (B2 of FIG. 5A). C of FIG. 5B is a radioradiogram of the reduced protein immobilized on the NC membrane. Horizontal arrows indicate the location of several abundant proteins that participate in the high molecular weight complex stabilized by S-S crosslinking. FIG. 5B is a western blot incubating C of FIG. 5A with mAb for β-tubulin prior to autoradiophotography. At least seven β-tubulin isoforms were immunostained at sperm protein extraction in the presence of DTT, followed by their expected molecular weight of 57 kDa, with a pI between 4.6 and 5.4. The position of β-tubulin in this magnetic radiograph is indicated by the vertical arrow in C of FIG. Β-tubulin itself was not radioiodinated by this method, even though the position of some tubulin isoforms in the radioradiograph was covered by luminescence from the surrounding iodide protein.

FIG. 6 shows protein imaging by reflected absorbance demonstrating that protein SP-10 in polymorphic tip body is not directionally iodinated. Radiolabeled proteins were solubilized with NP-40 and urea, separated by IEF / PAGE, transferred to NC paper and incubated with mAb MHS10. After colorimetric development with a DAB substrate, the SP-10 immunoreaction product was imaged on a magnetic radiograph [B] by reflection from an image enhancement screen, allowing the exact location of the SP-10 form to be visualized in shade. In the characteristic SP-10 pattern, the high molecular weight type is more acidic than the low molecular weight type, which is consistent with published information (32, 33). Sperm tip protein SP-10 contains three tyrosine residues and 12 histidine residues that can potentially be iodinated (32), but as long as the tip reaction sperm have been removed by post-label Percoll gradient centrifugation As not detected.

FIG. 7 is an autoradiograph of human sperm protein obtained from two donors directionally labeled and transferred to the NC membrane after two-dimensional electrophoresis, indicating that the pattern of iodide protein among donors has a high overall reproducibility. Shows. The same number of radioiodinated cells were solubilized from each donor and equal volumes of lysate were added to IEF / PAGE (A and C) and NEPHGE / PAGE (B and D). Certain iodide surface proteins, such as the 89-95 kDa group (vertical arrow), exhibited differences in relative concentrations as judged through integrated absorbance for the total density of all proteins on autoradiographs.

FIG. 8 shows NHS-LC-biotin, solubilized with NP-40 and urea, isolated via IEA / PAGE (A, C, E) or NEPHGE / PAGE (B, D, F) and transferred to NC membrane. Two-dimensional gel electrophoresis analysis of labeled human sperm protein. In A and B, biotinylated sperm protein was visualized with ECL and then incubated with AP-conjugated avidin. The non-biotinylated sperm proteins in C and D were only incubated with AP-avidin, resulting in five endogenous avidin-binding proteins with a molecular weight of 77 kDa and a pi of 6 to 6.5 (vertical arrow in C). The location of these endogenous avidin binding proteins in the surface biotinylation pattern is indicated by the same arrows in A, E and F. Biotinylated sperm protein was stained with colloidal gold and transferred to NC paper. In E, proteins were incubated with polyclonal antisern R-10 (produced for sperm surface hyaluronidase, a recombinant macaque PH-20). Three PH-20 protein spots with an apparent molecular weight of 53 kDa were immunolabeled (up arrow in E). 65 kDa antigen (acidic terminus of the gel) was stained with rabbit preimmune serum. Three 53 kDa spots were labeled with biotin (up arrow in A), which means that three forms of PH-20 can be surface biotinylated. In F, preimmune serum for PH-20 was used. Hollow arrow ( ) Indicates the position of basic 53 kDa on the NEPHGE gel. The 53 kDa form of PH-20 was not immunostained.

9 (FIGS. 9A and 9B) show the results of two-dimensional gel electrophoresis analysis of biotinylated human sperm protein solubilized with lysis buffer B (SDS, CHAPS, urea). 9A and 9B are proteins visualized by silver staining, and C and D are biotinylated proteins electrophoresed with NC paper after visualization with AP-avidin and ECL. 9B is an immunoblot showing the position of β-tubulin detected by incubation with mAb TU27. Are stained and the position of β-tubulin in the biotinylated two-dimensional patterns (A and C) is indicated by the same downward arrow. Hollow arrow in Figure 9A ( The high molecular weight complex indicated by) was immunoblotted with mAb and proved to contain β-tubulin. Neither α-tubulin nor β-tubulin was biotinylated. 9B is an immunoblot showing the fibrous herb antigen detected by mAb S69. The location of the fiber sheath component in the silver stained and biotinylated two-dimensional pattern is indicated by the same oblique arrows in B and D of FIG. 9A. Fibrous protein was not labeled with biotin. G and H in FIG. 9B are immunoblots of sperm surface hyaluronidase PH-20. Two isoforms of PH-20 were immunostained at 64 kDa and 53 kDa. When expanded to 53 kDa, it could be resolved into four isoforms. The strongest biotinylation occurred in the most acidic isoform of the 53 kDa component of PH-20. Weak immunoreactive spots of 65 kDa (indicated by oblique upward arrow heads in G of FIG. 9B and arrows in A and E of FIG. 8) were also present in the rabbit preimmune control (data not shown).

10 is an immunoblot using mAb RC-20 showing sperm protein phosphorylated at tyrosine residues. Human sperm fresh was solubilized in Lysis Buffer A, separated by IEF PAGE, and transferred to PVDF membrane. The predominant phosphorylated protein had a molecular weight of 89-95 kDa and a pi of 5.5-5.8. In addition, several weaker stained proteins can be observed. Arrowheads indicate five groups of directionally labeled phosphorylated proteins (see Table 1).

FIG. 11 shows a computer synthesized image of 1397 human spermatogenic protein that can be detected by silver staining after NP-40 / urea solubilization. Of the 98 proteins labeled by both surface iodide and surface biotinylation, 94 correspond to silver stained proteins and are circled. The characteristics of these proteins are shown in Table 1. This table is referred to as the Sperm Surface Index.

FIG. 12 shows 63 kDa sperm protein (pI 4.3) selected for the design of complementary and anti-complementary oligonucleotides (SEQ ID NOs: 16-18, 12, 19-20) via Edman degradation and tandem mass spectrometry. The obtained amino acid microsequence data is shown. From this microsequence pool, degenerate oligonucleotide primers were synthesized and PCR was performed (see FIG. 13).

FIG. 13 shows a PCR product obtained by amplifying human testicular RNA using degenerate oligonucleotides derived from microsequence data of 63 kDa, pi 4.3 protein, revealing one prominent PCR product at approximately 400 bp.

14 shows DNA sequences obtained from clones derived by RT-PCR using optimal degenerate primers. Numbers indicate the position of base pairs in the sequenced clones and the Calreticulin gene. "C" following the number indicates RT-PCR clone (SEQ ID NO: 25) and "H" indicates human Calreticulin cDNA (SEQ ID NO: 26) obtained from GenBank.

15 (FIGS. 15A and 15B) show microsequences and complementary and reverse complementary oligonucleotides (SEQ ID NOS 27-40) obtained from tandem mass spectrometry for sperm surface protein I-23.

16 shows the DNA sequence (SEQ ID NO: 41) encoding a portion of sperm surface protein I-23. This DNA sequence was obtained by sequencing a PCR product obtained by amplifying human testicular DNA using a pool of degenerate primers synthesized based on the microsequence shown in FIG. 15.

FIG. 17 is an Encyclopedia of 967 silver stained human sperm proteins with an isoelectric point range of 3.5-> 6.5, obtained by laser scanning and computer digitization of two-dimensional electrophoresis with isoelectric focusing.

FIG. 18 is a dictionary of 435 silver stained human sperm proteins having an isoelectric point range of 6.5-> 10.5, obtained by laser scanning and computer digitization of two-dimensional electrophoresis with a non-equilibrium pH gradient of first dimension.

Detailed Description of the Preferred Embodiments

According to the present invention, conventional molecular biology, microbiology, protein chemistry, and DNA recombination techniques can be used, which are within the skill of the art. Such techniques are explained fully in the literature. Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, RM, ed. (1994)]; "Cell biology: A Laboratory Handbook" Volumes I-III [JE Celis ed (1994)); "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985); "Transcription And Translation" [B. D. Hames & S. J. Higgins, eds. (1984); "Animal Cell Culture" [R. I. Freshney, ed. (1986); "Immobilized Cells And Enzymes" [IRL Press (1986)]; See B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore, as used herein, the following terms will follow the definitions set forth below.

A "replicon" is any genetic component (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, ie that can replicate under its control.

A "vector" is a replicon, such as a plasmid, phage or cosmid, to which other DNA fragments are attached, which can cause replication of the attached fragments.

"DNA molecule" refers to a polymer form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) that is single stranded or double stranded. The term refers only to the primary and secondary structures of the molecule and does not limit the structure of the molecule to any particular tertiary form. The term therefore encompasses, in particular, double stranded DNA found in linear DNA molecules (eg restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence can be provided according to the normal practice of providing only sequences in the 5 'to 3' direction along the nontranscribed strand of DNA (strands with sequences homologous to the mRNA). .

"Origin of replication" refers to those DNA sequences that participate in DNA synthesis.

A DNA “coding sequence” is a double stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The edge of the coding sequence is determined by the start codon at the 5 '(amino) terminus and the translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, prokaryotic sequences, cDNAs from eukaryotic mRNAs, genomic DNA sequences from eukaryotic (eg mammalian) DNAs, and synthetic DNA sequences. The polyadenylation signal and transcription termination sequence will typically be located 3 'of the coding sequence.

Transcriptional and translational regulatory sequences are DNA regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, and the like, and are required for the expression of coding sequences in host cells.

A "promoter sequence" is a DNA regulatory region that can bind RNA polymerase within a cell to initiate transcription of a downstream (3 'direction) coding sequence. To define the present invention, the promoter sequence abuts its transcription start position at its 3 'end and extends upstream (in the 5' direction) such that the minimum number of bases required to initiate transcription at detectable levels above the background and Contains elements. Within the promoter sequence are the protein initiation site (consensus sequence) that binds the RNA polymerase as well as the transcription initiation site (which is conveniently defined by mapping with nuclease S1). Eukaryotic promoters usually, but not always, include a "TATA" box and a "CAT" box. Prokaryotic promoters include a shine-dalgarno sequence and -10 and -35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of other DNA sequences. The coding sequence is "under control" of the transcriptional and translational regulatory sequences within the cell when the RNA polymerase transcribes this coding sequence into mRNA (which is subsequently translated into a protein encoded by the coding sequence).

The "signal sequence" can be included before the coding sequence. The sequence encodes a signal peptide at the N-terminus of the polypeptide, which communicates with the host cell to either direct the polypeptide to the cell surface or secrete the polypeptide into the medium, which causes the protein to bind the cell. It is cut by the host cell before leaving. Signal sequences can be found to bind various proteins inherent in prokaryotes and eukaryotes.

The term "oligonucleotide" as used herein to refer to a probe of the present invention is defined as a molecule consisting of two or more ribo or deoxy nucleotides, preferably consisting of three or more. Its exact size will depend on many factors, that is to say on the ultimate function and use of the oligonucleotide.

As used herein, the term “primer”, whether naturally occurring or produced synthetically through purified restriction digestion, complements any nucleic acid strand in the presence of nucleotides and inducing agents such as DNA polymerase at appropriate temperatures and pH. Refers to oligonucleotides that can serve as a starting point for synthesis in conditions under which synthesis of a primer extension product can be induced. The primer may be single stranded or double stranded and should be long enough to initiate the synthesis of the desired extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, use of the method, and the like. For example, if for diagnostic purposes, depending on the complexity of the target sequence, oligonucleotide primers may include fewer nucleotides but typically include 15 to 25 or more nucleotides.

Primers herein are selected to be "substantially" complementary to other strands of a particular target DNA sequence. This means that the primer must be sufficiently complementary to hybridize with its counter strand. Thus, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5 'end of the primer and the rest of the primer sequence can be complementary to its strand. Alternatively, non-complementary bases or longer sequences may be intersperse in the primers, provided that the primer sequences are sufficiently complementary to the sequences of the strands that hybridize with them, forming a template for the synthesis of extension products.

As used herein, the terms "limiting endonuclease" and "limiting enzyme" refer to bacterial enzymes that cleave double stranded DNA at or near specific nucleotide sequences.

The cell was "transformed" by such DNA when exogenous or heterologous DNA was introduced into the cell. The transforming DNA may or may not be integrated (by covalent bonds) into the chromosomal DNA making up the genome of the cell. In prokaryotes, yeast and mammalian cells, for example, the transforming DNA can be maintained on episomal elements such as plasmids. In the case of eukaryotes, stably transformed cells refer to cells in which the transformed DNA is integrated in the chromosome and inherited into daughter cells through chromosomal replication. This stability is evidenced by the ability of eukaryotic cells to establish cell lines or clones of daughter cell populations containing transformed DNA. A "clone" is a group of cells derived through mitosis from one cell or a common ancestor. "Cell line" refers to a clone of primary cells that can stably propagate in vitro for generations.

Two amino acid sequences "substantially homologous" means that at least about 75% (preferably at least about 80%, most preferably at least about 90 or 95%) of nucleotides match the defined length of the DNA sequence. I mean. Substantially homologous sequences can be found by using standard software available in the sequence data bank or by comparison, for example, through Southern hybridization experiments under stringent conditions defined for a particular system. Defining appropriate hybridization conditions is known in the art (Maniatis et al., Previous book; DNA Cloning, Vols. I & II, previous book; Nucleic Acid Hybridization, see above).

"Substantially homologous" of two amino acid sequences indicates that at least about 70% (preferably at least about 80%, most preferably at least about 90 or 95%) of amino acid residues are identical or conservative substitutions. Means that.

A “heterologous” region in a DNA construct is a piece of identifiable DNA that is within a larger DNA molecule, which in nature is not found with the large DNA molecule. Thus, if a heterologous region encodes a mammalian gene, the gene will typically attach laterally to DNA that is not flanked by this mammalian genomic DNA within the genome of the source organism. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (eg, a cDNA whose genomic coding sequence comprises an intron, or a synthetic sequence having a codon different from the native gene). Allelic variations or naturally occurring mutations will not result in heterologous regions of DNA as defined herein.

An "antibody" is any immunoglobulin that binds to a specific epitope, including antibodies and fragments thereof. This term encompasses polyclonal, monoclonal, and chimeric antibodies, which are described in more detail in US Pat. No. 4,816,397 and US Pat. No. 4,816,567.

An "antibody combining site" is a structural site of an antibody molecule consisting of heavy and light chain variable and hypervariable regions that specifically bind to an antigen.

As used herein in various grammatical forms, an “antibody molecule” is intended for both the immunologically active site of an immunoglobulin molecule and an intact immunoglobulin molecule.

Examples of antibody molecules are portions of immunoglobulin molecules, including intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and paratopes, wherein paratopranes (Fab, Fab ', F (ab') ₂ and F encompassing those known in the art such as (v) and the like, which are preferred for use in the diagnostic and therapeutic methods herein.

Fab and F (ab ') ₂ portions of antibody molecules are prepared by well known methods by proteolytic reactions of papain and pepsin, respectively, on substantially intact antibody molecules. See, eg, US Patent No. 4,342,566 to Theofilopolous et al. Fab 'antibody molecule moieties are also well known, using mercaptoethanol to reduce disulfide bonds linking the two heavy chain moieties, and the resulting protein mercaptans are alkylated using reagents such as iodoacetamide to form F (ab'). ) Is prepared from ₂ parts. Antibodies comprising intact antibody molecules are preferred herein.

As used in various grammatical forms, "monoclonal antibody" refers to an antibody having only one type of antibody binding site capable of causing an immune response with a particular antigen. Thus, monoclonal antibodies show a single response affinity for any antigen that immunes to them. Thus, monoclonal antibodies can include antibody molecules, such as bispecific (chimeric) monoclonal antibodies, that have multiple antibody binding sites that are immunospecific for different antigens per antibody binding site.

The term “pharmaceutically acceptable” refers to the molecule itself and to a composition that is physiologically resistant and which, when administered to a human, does not cause an allergic reaction or similar inappropriate reaction, such as gastrointestinal upset, dizziness, and the like.

A "therapeutically effective amount" is a characteristic of a clinically significant change in S phase activity of a target cell mass, or other condition that may be accompanied by the presence and activity of the condition, such as, for example, elevated blood pressure, fever or white blood cell count. Sufficient amount to prevent or preferably reduce by at least about 30%, more preferably at least about 50% and most preferably at least about 90%.

When a DNA sequence is "operably linked" to an expression control sequence, it is meant when the expression control sequence controls and regulates the transcription and translation of that DNA. The term "operably linked" means that there is an appropriate starting signal (eg ATG) in front of the DNA sequence to be expressed, the DNA sequence being expressed under the control of an expression control sequence to produce the desired product encoded by the DNA sequence. To ensure that the correct reading frame is maintained. If the gene that needs to be inserted into a recombinant DNA molecule does not contain an appropriate starting signal, such starting signal can be inserted before the gene.

The term “standard hybridization conditions” refers to salt and temperature conditions substantially equivalent to 5 × SSC and 65 ° C. for both hybridization and washing. However, those skilled in the art will appreciate that such "standard hybridization conditions" will vary depending on the specific conditions such as the concentration of sodium and magnesium in the buffer, the length and concentration of the nucleotide sequence, mismatch (%), formamide (%), and the like. Will understand. Also important in the determination of "standard hybridization conditions" is whether the two sequences to hybridize are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are readily determined by those skilled in the art according to well-known formulas, where hybridization is usually performed below 10-20 ° C. and washed to greater stringency than the predicted or measured T _m .

Detailed knowledge of sperm surface molecules not only helps to understand the complex process of differentiation and maturation, but also because cell membranes are critically important for sperm function.

The combination of directed labeling and detergent solubilization in the method of the present invention allows for the analysis of relatively few cells, bacteria or viruses, and specific proteins (physiologically or experimentally derived) to be studied in one individual or sample. Daily changes in the pattern are possible. In addition, directional labeling technology provides information related to the exofacial orientation of plasma membrane proteins, which cannot be achieved by nitrogen cavitation technology alone.

First, acidic, neutral and basic human sperm proteins are all analyzed in a single way and the two methods for labeling surface exposed proteins are compared. A comprehensive 2D database can be established for membrane surface proteins, particularly human sperm proteins.

One way to immediately use a deep understanding of the composition of sperm cell membranes is to design contraceptive vaccines. Knowledge of the molecules accessible by antibodies and T cell mediators present on the surface of biological targets provides a theoretical basis for developing vaccines. In the case of contraceptive vaccines based on sperm antigens, agglutination with sperm, immobilization of sperm, solubilization of sperm, or an immune response that interacts with surface accessible proteins prevents fertilization from occurring. If a woman develops antibodies against sperm surface proteins in the secretions of the cervix, uterus and fallopian tubes, sperm progression through the female reproductive tract will be hampered at various anatomical levels.

An ideal contraceptive vaccine based on sperm is a mixture of sperm antigens derived from all major domains of the sperm plasma membrane, including the head, center and tail, as well as the inner stromal membrane that forms a limiting membrane over the sperm head after sperm response. It may be planned to include (9). Monoclonal or polyclonal antibodies that block sperm function have led to the identification of several candidate sperm vaccine immunogens in the plasma membrane of sperm or bound to the inner tip membrane (10), but the full range of potential human sperm surface immunogens Has never been done before.

Two-dimensional gel electrophoresis developed by O'Farrell (11, 12), incorporated herein by reference, can be used in combination with gel computer analysis to analyze complex mixtures of polypeptides. Specialized computer software facilitates the quantitative analysis of two-dimensional gels, including the ability to compare various computer images, and provides detailed insight into changes in protein patterns caused by changing experimental manipulations or environmental stimuli (13-17).

The present invention performs major labeling by performing detergent extraction, two-dimensional electrophoresis, and computer image analysis after directional labeling. From information obtained using this method, cell surface protein dictionary indexes, membrane surface protein dictionaries, or sperm protein dictionaries can be obtained, which are numbered for various proteins (1397 total sperm proteins) and the proteins are coordinated (Mr and pI), and lists the "integral intensity" which is a measure of staining intensity for all other proteins.

Cell membranes and microbial membranes can be pulverized using any method known in the art, including detergent treatment, pulverization using motors and pestles, sonication, and the like. Cleaning agents may include Nonidet P-40 (NP-40), Tween, SDS, and the like. Among other things, denaturants such as urea can be used.

Labeling can be performed using all labels suitable for labeling of surface proteins. Examples of labels are iodine (I ¹²⁵ ) and biotin. A "primary target" for further analysis, such as sequencing, is a protein that is preferably labeled using one or more methods.

Methods of separating proteins using two-dimensional gel electrophoresis are well known in the art. Preliminary two-dimensional gels can be run to obtain meaningful (sequencing) quantities of protein from the spots identified by gel analysis.

If necessary, protein separation by electrophoresis is performed by varying the concentration of ampoules in the first dimension, which allows the limited area of the two-dimensional map to be expanded to fill the entire two-dimensional gel form. This enables enlarged separation of proteins within a given pI and Mr spectral region, thus increasing resolution in areas where many proteins are clustered, and also allowing large amounts of protein loading to optimize yields. Proteins are stained with Ponceau, Coomassie, Silver or Gold, depending on the characteristics of each protein and its future use. After electrophoresis on a two-dimensional gel, the proteins are electroblotted with membrane support and digested on acrylamide gels with trypsin or lysyl peptidase to obtain peptides for use in Edman degradation or tandem mass spectrometry.

Databases for surface proteins can be used as a reference for subsequent cell membrane protein analysis and can also guide target selection for microsequencing and antibody preparation. Antibodies that are candidates for microsequencing include those that react with the serum of a patient who has undergone infertility or vasectomy. Another primary microsequencing candidate is sperm aggregation antigen 1 [SAGA-1]. MES-8 mAb aggregates with 100% of sperm in human semen, impedes sperm invasion in cervical mucus, inhibits human sperm from invading hamster eggs, and modulates kin sperm in vitro in mouse eggs. Inhibits human sperm binding to human zona pellucida by up to 70%. SAGA-1 antigen is located on the entire sperm surface.

Affinity purification methods can also be used to collect desired proteins that are present in relatively less abundance. Washed and harvested cells, bacteria or viruses can be directionally labeled with NHS-SS-biotin (or NHSO iminobiotin), wherein biotinylation probes the reducing disulfide. Proteins are solubilized in non-reducing conditions and biotinylated surface complexes precipitate with beads encoded with immobilized streptavidin. The precipitated protein can be eluted through reduction and washing and analyzed by two-dimensional electrophoresis. Microsequencing of biotinylated surface proteins integrated with this affinity bead method can be undertaken from heavily loaded preparative two-dimensional gels or after reverse phase HPLC separation.

Sequencing of protein spots is performed by any method known in the art. Preferably it is carried out via Edman decomposition and mass spectrometry, preferably tandem mass spectrometry (TMS).

Coomassie stained protein spots on PVDF membranes were loaded into blot cartridges for sequencing. To obtain internal sequences, proteins are digested in gels, especially with lysyl endopeptidase or other proteases, and peptides are extracted and separated into columns, in particular 1 mm diameter C18 columns. The mass and purity of the isolated peptide is determined by the matrix assisted laser emission and ionization time of flight mass spectrometry using Finnigan LaserMAT. After coating with polybrene, a solution containing the desired isolated peptide can be applied to a glass fiber filter to which a condition cycle is applied. The NH-terminal amino acid sequence of this peptide is determined by protein sequencing. Sequencing of internal peptides can be performed with 20 to 30 cycles of Edman degradation of intact (whole) proteins, provided that they are appropriate when judged based on the mass of the protein calculated by mass spectrometry. The sequencing method derivatizes the NH-terminal amino acid with phenylisothiocyanate, and then cleaves with trifluoroacetic acid to obtain the anilinothiozolinone amino acid, which is then used with an acid to form a phenylthiohydrantoin (PTH) amino acid. Standard Edman chemistry can be used. PTH amino acids can be identified and quantified using Applied Biosystems 140 HPLC, especially 2.1 mm diameter C18 columns optimized for PTH amino acid analysis.

High sensitivity internal sequencing can be performed by tandem mass spectrometry. Protein spots are reduced, alkylated in polyacrylamide gels and then treated with protease (usually trypsin) in ammonium bicarbonate. Peptides obtained by this method are extracted from polyacrylamide, in particular with 50% acetonitrile / 5% formic acid, and then the extract is concentrated to almost dryness and redissolved to make it intact, preferably using 1% acetic acid. do. Peptides were connected to an LC-electrospray mass spectrometer, in particular 75 gm i.d. connected to Finnigan MAT TSQ7000 electrospray ionization-tandem quadrupole mass spectrometer. Analysis can be done using capillary HPLC columns. Analyzing these results can determine the molecular weight of the peptide and the information used to replan the tandem mass spectrometry for analysis of dissociation (CAD) activated by collisions of specific peptides. In CAD experiments, the ions of a given haze ratio (m / z) are the mass selected by the first mass spectrometer. This method selects peptides for sequencing based on their molecular weight to effectively remove all other peptides from the analysis. The selected peptide is collided with an element of argon to fragment, and the product of this fragmentation reaction is subjected to mass spectrometry with a second mass spectrometer of a tandem mass spectrometer. This gives a CAD mass spectrum from the amino acid sequence of the peptide. This method is repeated under computer control to obtain sequence information for each peptide in the digest.

Sequencing by mass spectrometry is very sensitive and internal sequence data can be obtained from samples containing 1-10 pm starting material. Using a first mass filter of a tandem mass spectrometer, several peptides resulting from digesting the protein are isolated, thus avoiding the chromatographic steps necessary to obtain pure peptides in the Edman sequencing method. However, Edman sequencing obtains data from the N terminus of the intact protein (if the NTH terminus is not blocked), resulting in 30 or more amino acid residues in some cases. Although less sensitive than mass spectrometry sequencing, Edman digestion can analyze sequences of peptides longer than mass spectrometers and easily distinguish between leucine and isoleucine, which are not well distinguished by mass spectrometry. Since the Edman method requires purification of the peptide before loading it into the instrument, it is necessary to carefully separate and concentrate the protein by preliminary two-dimensional electrophoresis. The Edman method requires approximately 10-15 pmole of starting material to obtain an internal sequence of sufficient length to design the oligonucleotide. However, amounts as low as 2 pmole are sufficient for long amino terminal sequence analysis of full length proteins.

From the obtained amino acid sequence information, oligonucleotides can be designed, preferably degenerate oligonucleotides representing all possible codon combinations of that sequence (otherwise using inosine at the third position in the codon), such oligonucleotides Can be cloned by hybridization to a library containing cDNA encoding membrane surface proteins, or used to amplify by polymerase chain reaction (PCR), preferably RT-PCR.

The RT-PCR method using two anchoring primers takes advantage of the simplicity and speed of the PCR protocol. However, since the genetic code is degenerate, the selection of oligonucleotides based on known amino acid sequences will require the synthesis of many probes corresponding to a given amino acid sequence.

At least two amino acid sequences of seven or more residues in length are required from each protein (however, if two sequences are not obtained, one sequence can be used with the 5 'and 3' RACEs). The optimal primer length for PCR is 20 to 30 nucleotides. To do this, in order to design two primers of at least 20 bases in length, a clear sequence of at least seven contiguous amino acids must be obtained from at least two positions in the primary structure of a given protein.

Detect which part of a given amino acid sequence will be selected for targeting and the exact composition of each oligonucleotide, including the GC content of the assumed oligonucleotide, the number of distinct amino acid residues sequenced, the melting point inherent in the assumed oligonucleotide pool Consider a combination of factors. In order to prepare oligonucleotides that fall within an optimal size of 20-30 bases in length, a minimum stretch of at least 7 amino acids must be obtained by microsequencing. If longer amino acid sequences can be obtained, there will be many opportunities for optimal oligonucleotide design. To facilitate RT-PCR of cDNA fragments of appropriate length, oligonucleotide pools may be prepared from independent regions of the RNA to be amplified, via both N-terminal and internal protein sequences or internal amino acid sequences. The probe can be used to amplify a cDNA fragment in several ways: (1) using two sets of anchored oligonucleotides in the RT-PCR reaction, which can then be used to screen the cDNA library for full length cDNA. Method of obtaining, (2) adjusting the PCR-RACE reaction using one or more sets of oligonucleotides to amplify the 3 'or 5' cDNA fragments and using the cDNA fragments to screen the cDNA library, (3) PCR and PCR -RACE can be used in combination to obtain full-length cDNA by binding sequences from separate PCR reactions together, and (4) to screen cDNA libraries directly using oligonucleotides.

When one amino acid sequence is obtained from an unknown protein, a modified RACE protocol can be performed using one "immobilized" optimal oligonucleotide pool to derive the 3 'or 5' sequence upstream or downstream of the known sequence. have. In this method, one internal amino acid sequence can be used to design anti-complementary oligonucleotides to a hypothesized sense (5'-RACE) or anti-cent (3-RACE) strand. The second oligonucleotide to modulate the amplification reaction is derived from the known sequence of the linker at the 5 'or 3' end of a commercial cDNA library. This method is currently available in kit form (Marathon-Ready cDNAS ™, Clonetech).

Suitable cDNA libraries include (1) 5'-stretch (Clontech), (2) λ-zap and (3) λ-gt 11 cDNA libraries. All three libraries have been used to obtain the full length ORF of various testicular proteins. Methods of probing libraries using amplified cDNA probes are well described in the literature. In short, oligonucleotides can be used for labeling, purifying and hybridizing using (λ- ³² P) -rATP and polynucleotide kinases as directed by the manufacturer. As a positive control, northern blots can be probed to determine whether the PCR product will recognize specific mRNA messages within the total RNA. The 5'-stretch cDNA library (Clonetech) is plated on 150 mm agar according to the manufacturer's instructions and transferred twice with MSI filter (Fisher). This filter can then be crosslinked with UV, prehybridized and hybridized overnight with a standard solution. The filter can then be exposed to the X-ray film and the resulting positive clones can be identified from a pair of filters to remove false positives. The film is then rearranged with the original culture plate and the core is taken. The primary plaques can be trialed and rescreened until all plaques on a given plate provide a positive response. In each step of purification the phage sample should be kept at -20 ° C. Once isolated, the clones are amplified by PCR, using PCR prepared primers for phage sequences proximal to the cloning position used in library preparation. The cDNA insert amplified by PCR can be cut with enzyme and the resulting fragments can be isolated and sized on an agarose gel. Restriction analysis of separate clones can produce fragments of similar size, which can confirm that the degenerate oligonucleotides recognize the same clone.

Degenerate oligonucleotides can be used to screen cDNA libraries directly, but usually only if a PCR-based approach has not been performed. Mixtures of ³² P terminal labeled oligonucleotides can be selected based on systematically staggered codons encoding amino acid microsequences. Oligonucleotides are designed according to the above parameters with an emphasis on developing pools with as low degeneracy as possible (<300). Positive results were obtained from oligonucleotide pools of 17-oligomer. If the oligonucleotide pools are larger than this, several pools containing all possible combinations can be used. First control experiment, first the terminal cover puul poly ^- (A) - by hybridization to the Northern blot containing mRNA, can determine the hybridization conditions indicates the cleaning and positive band when the X-ray film exposed. Once hybridization and wash conditions (salts and temperatures) are established, the cDNA libraries listed above are repeatedly plated, lifted and probed with labeled oligonucleotide pools at the conditions suggested by Ausubel. Can be. The double lifts can be compared to eliminate false positives and secondary and tertiary screening can be performed to ensure the purity and accuracy of the estimated clones. If possible, the same library lift can be rescreened with a second oligonucleotide mixture derived from another region of the unknown protein.

In addition, expression libraries can be screened using antibodies to membrane surface proteins.

Monospecific antiserum may be generated for protein spots isolated from two-dimensional gels. Two methods can be taken: (1) emulsifying the acrylamide gel core containing the protein in Freund's complete adjuvant to immunize the animal according to standard intramuscular immunization, or (2) Including nitrocellulose pieces may be immersed in Freund's complete adjuvant and then implanted subcutaneously and intraperitoneally. Aliquots of the monospecific sera produced by these methods can be determined by Western blot to determine the end point titer and the specificity for the desired protein spots. If the appropriate concentration of a given antiserum is low, the antibody enhancement solution can be made by affinity purification of the antibody using Olmstead technology using nitrocellulose binding proteins from specific protein bands or spots. The expression library was plated to a sufficient number (6 times the density used for the nucleotide probe) to reveal all three reading frames in both directions, propagated at 37 ° C. for 3 to 4 hours, followed by a filter soaked in 10 mM IPTG. And incubation at 42 ° C. Monospecific and pre-immune serum obtained from the same animal can be adsorbed twice at 4 ° C. overnight with E. coli bound to cyanogen bromide activated Sepharose to remove reactivity with E. coli protein.

Restriction analysis then converts the cDNA bearing phage clones containing the largest inserts into plasmid form. Highly purified plasmid DNA required for accurate sequencing of long stretches can be prepared by amplifying plasmid-bearing bacteria into large scale (≧ 100 ml) cultures followed by CsCl equilibrium centrifugation by standard methods. Standard primers for plasmid sequences except for the cloning positions are prepared and used for sequencing. Sequence analysis is preferably carried out by dideoxy chain termination methods using a Sequenase® kit. Sequence analysis proceeds in both directions, producing new cDNA specific primers for the internal sequence and continuing the sequence analysis until the sequences overlap. Alternatively, four color sequencing can be performed with Applied Biosystems Prism 377 automated DNA sequencing. An open reading frame can be obtained by computer analysis and estimation start point to confirm that the clone is full length. In addition, the original sequence of amino acids obtained through microsequencing should be identified.

If the sequence cannot be derived to provide a cDNA clone that is a "doubleanchored" RT-PCR derivative, oligo-d (T) can be used as the second "immobilized" oligonucleotide.

Sometimes clones in some areas have unclear consequences of compression. In this case, a readable sequence can be obtained using TaqTrack® Sequencing Kit (Promega), which performs the sequencing reaction at 72 ° C rather than 37 ° C. Higher temperatures in the reaction step result in more reliable melting of any potential single stranded region, resulting in a reliable clear sequence.

Sequences can also be compared to the genetic data bank to identify identity and homology with known proteins.

To assess putative tissue specificity of antigens in human and primate models, RNA northern blot analysis from major organs of monkeys and humans can be used. Total RNA can be isolated from various tissues stored at -70 ° C. Poly (A +) RNA is purified from total RNA by oligo-d (T) chromatography. The RNA can be concentrated by ethanol precipitation and then resuspended in sterile distilled water (105 ml). The amount of poly A + RNA in each sample can be quantified by reading its absorbance at 260 nm [OD 1.0 = 40 mg / ml]. Equal amounts of poly A + RNA samples from each tissue to be tested are separated on denatured agarose / formaldehyde gels and the RNA is transferred to nylon. After washing this nylon with 2 X SSC for 5 minutes, RNA is crosslinked by UV light with nylon. The nylon is prehybridized and then hybridized overnight at 65 ° C. in fresh hybridization buffer containing 10 mg / ml of probe labeled with ³² P-dCTP. Probes may consist of cDNA for antigen under study or control probes of β-actin or cyclophylline. Probes can be labeled with random priming using kits and protocols purchased from Promega. If necessary, both low and high stringency conditions should be tested. Self-radiographs are exposed using a sensitivity-enhanced screen. After hybridization with a test probe, the nylon blot is peeled off and reprobed with one of the controls (β-actin or cyclophylline) to test for the same drop of RNA.

Tissue specificity of the antigen can be assessed by amplifying the cDNA resulting from reverse transcription of RNA from each tissue by polymerase chain reaction (PCR). Primers specific to the RNA of interest can be used, corresponding to the initiation and termination of each translation. Positive controls may use β-actin specific primers that are spaced apart from each other by intermediary sequences of 198 nucleotides, selected based on conserved regions in the β-actin cDNA. The PCR product is visualized, stained with ethidium bromide, and the size is evaluated. Whether the PCR product actually corresponds to the desired material can be determined by hybridizing in Southern blot with cDNA specific for the antigen under study.

Tissue specificity of the antigen can be assessed using another method by immunohistochemical studies, preferably using affinity purified antigens. Paraffin-impregnated tissue layers can be collected and stored. Proteins can be placed in the tissue layer by immunoperoxidase labeling of paraffin-impregnated tissue sections. Tissue can be fixed with 10% neutral buffered formalin (Sigma) for several hours. The tissues are dehydrated in ethanol series, impregnated with paraffin, excised and placed on slides. Prior to use, tissue sections are dewaxed, rehydrated and treated with 0.25% hydrogen peroxide to block endogenous peroxidase activity. Nonspecific protein binding sites can be blocked by incubating the slides in PBS containing 5% normal goat serum (NGS). Slides were incubated with affinity purified rabbit polyclonal antibodies generated against diluted recombinant protein in PBS-NGS or control preimmune serum, followed by peroxidase-conjugated goat anti-rabbit IgG / IgM in PBS-NGS. Incubate with. Slides can be washed with PBS and immunoreactive proteins can be visualized by staining with TrueBlue peroxidase substrate (KPL) or gold enhanced staining. The presence of blue or black precipitates indicates the immunoreactive protein (s).

In sperm surface protein microsequence analysis performed so far, heat shock protein 90 alpha (82%), gastrin binding protein (94.7%), human calreticulin (15 amino acids and 100%), mouse fibrous protein ( 64%) and serum amyloid component precursors were found.

Approximately 40% of the microsequenced proteins appear to be new.

Table I shows the coordinates of the novel sperm surface proteins.

Microanalyzed surface protein obtained from sperm dictionary (see Table 11 for additional surface proteins) Protein coordinates Mw, pI Sample (1 = protein immobilized on PVDF membrane, 2 = peptide from gel digest) Directional Labels (I = 125-I, B = Biotin) Sequencing Method Residue obtained ^* Data analysis 92.5 kDa, 5.3 2 I / B TMS 12 seq., 5-12 aa New surface 92 kDa, 5.6 2 I / B E 9 aa New surface 90 kDa, 5.7 2 I / B TMS 2 seq., 7-10 aa New surface 90 kDa, 4.9 One I / B E 6aa New surface 88 kDa, 4.0 2 I / B TMS 6 seq., 5-10 aa New surface 32 kDa, 5.2 2 I / B TMS 3 seq., 8-9 aa New surface E = Edman degradation, TMS = tandem mass spectrometry, * = number of peptide sequences obtained, range of sequence length

Membrane proteins identified by the methods of the invention have both diagnostic and therapeutic uses.

If it is necessary to reduce or inhibit the binding of membrane surface proteins to any cell or other target, the interaction of membrane surface proteins with their ligands or receptors may be introduced by introducing appropriate inhibitors, including antibodies to these membrane surface proteins. You can block the action.

Agents that antagonize or mimic membrane surface proteins, or that exhibit control over the production of these proteins, may be administered to patients suffering from deleterious medical conditions accompanied by the presence of target cells, bacteria, or viruses. It may be prepared in a pharmaceutical composition containing a suitable carrier at a concentration effective for administration. Parenteral methods such as subcutaneous, intravenous and intraperitoneal infusions, catheterization and the like can be used in various ways of administration. The average amount of membrane surface protein or subunits thereof may vary, and should, in particular, be advised and prescribed by physicians and veterinarians.

In a preferred embodiment, the invention relates to the administration of membrane surface proteins or antibodies thereto as contraceptives to inhibit sperm cell and egg interactions, to inhibit sperm transport or to fix sperm in the female reproductive tract.

Antibodies, including membrane surface proteins and both polyclonal and monoclonal antibodies, may have specific diagnostic uses, for example to detect and / or measure conditions such as bacterial or viral infections, immunity to sperm cells, and the like. Can be used for For example, using membrane surface proteins or subunits thereof, polyclonal and monoclonal antibodies against them may be used in various cell media, for example hybridoma technology using fused mouse splenic lymphocytes and myeloma cells, and the like. It can manufacture by a well-known technique. Similarly, small molecules can be found or synthesized that mimic or antagonize the activity of the membrane surface proteins of the invention and can be used in diagnostic and / or therapeutic protocols.

As an alternative to generating polyclonal antibodies against the whole protein, sufficient amino acid sequences can be obtained through microsequencing to develop synthetic peptide immunogens. These peptide immunogens can be used as immunocontraceptors. Alternatively, antipeptide antiserum can be generated against chimeric peptides unless cloning of the desired protein is performed using direct library probes using RT-PCR or oligonucleotides, or polyclonal antibodies prepared as above. In this strategy, amino acid sequences can be conjugated using a GPSL (SEQ ID NO: 2) intervening linker to a harmonized T cell epitope obtained from tetanus toxin (VDDALRNSTKIYSYFPSV (SEQ ID NO: 1)). Antipeptide antisera can then be used to screen cDNA expression libraries.

To obtain polyclonal antibodies against the recombinant antigens, New Zealand white rabbit females were injected with a total of six injections of recombinant antigen (about 500 μg) dissolved in complete (one) and incomplete (five) Freund's adjuvant at three week intervals. can do. Serum can be obtained weekly after two injections.

To obtain affinity purified antibodies, brominated cyanogen activated sepharose (Sigma Chemical Co., St. Louis, MO) can be used as a stationary phase for purified recombinant antigens. The polyclonal antibody is placed in an antigen affinity column to allow binding of the antibody. Bound antibody is eluted and the UV absorbance of the fractions is observed. The fractions are collected and then blotted with anti-rabbit Ig reagent to reveal bands representing purified Ig. Retention of antigen binding can be demonstrated by subjecting several batches of ELISA endpoint titration of purified IgG to the same protein concentration for a given amount of recombinant antigen.

In the study of sperm antigens, affinity purified antibodies to recombinant sperm antigens can be immunized with Western blots of two-dimensional gels loaded with sperm membrane extracts.

Sperm function analysis can be performed to assess whether affinity purified antibodies to recombinant antigens affect sperm function (s) by reacting with native antigens on the sperm surface. These assays include the identification of antigens on sperm by immunofluorescence, the determination of surface location by FACS, sperm / oval testing [sperm invasion test] and sperm / translucent interaction [translucent band analysis], sperm aggregation and immobilization. May be included.

The general method of preparing monoclonal antibodies using hybridomas is well known. Immortal antibody producing cell lines may also be prepared by techniques other than fusion, such as by directly transforming B lymphocytes with carcinogenic DNA or by transfecting with Epstein-Barr virus. See, eg, M. Schreier et al., "Hybridoma Techniques" (1980), (Hammerling et al., "Monoclonal Antibodies And T-cell Hybridomas" (1981)), (Kennett et al., "Monoclonal Antibodies"). (1980)). See also US Pat. Nos. 4,341,761, 4,399,121, 4,427,783, 4,444,887, 4,451,570, 4,466,917, 4,472,500, 4,491,632, 4,493,890.

Plates of monoclonal antibodies generated against membrane surface peptides can be screened for various properties such as isotypes, epitopes, affinity, and the like. There is a particular need for monoclonal antibodies that neutralize the activity of membrane surface proteins or subunits thereof. High affinity antibodies are also useful when immunoaffinity purification of natural or recombinant membrane surface proteins is possible.

Preferably the antibody used in the diagnostic methods of the invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). It is also preferred that the antibody molecule as used herein is in the form of a Fab, Fab ', F (ab') ₂ , or F (v) portion of the entire antibody molecule.

As suggested above, the diagnostic methods of the present invention employ various assays that include an effective amount of a binding partner for a membrane surface protein, eg, an antibody, preferably an affinity purified polyclonal antibody, more preferably mAb. Examining the cell or serum sample or medium. It is also preferred that the antibody molecule used herein is in the form of a Fab, Fab ', F (ab') ₂ or F (v) moiety or the entire antibody molecule. As described above, patients who may benefit from this method include those who are suffering from a bacterial or viral infection or who are looking for contraception.

It is well known in the art how to isolate membrane surface proteins, induce antibodies, and optimize the ability of antibodies to assist in the testing of target cells. Methods of making polyclonal anti-polypeptide antibodies are well known in the art. See US Pat. No. 4,493,795 to Nestor et al. Monoclonal antibodies, typically monoclonal antibodies comprising Fab and / or F (ab ′) ₂ portions of useful antibody molecules, are herein incorporated by reference (Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York, 1988. Briefly describe myeloma or other to prepare hybridomas that produce monoclonal antibody compositions. Self-perpetuating cell lines are fused with lymphocytes obtained from the spleen of a mammal overimmunized with a membrane surface protein or binding portion thereof.

Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HATs. Hybridomas producing monoclonal antibodies useful in the practice of the present invention are identified by their ability to immunoreact with membrane surface proteins and their specific infective activity or their ability to inhibit fertilization of target cells.

Monoclonal antibodies useful in the practice of the present invention can be prepared by initiating monoclonal hybridoma cultures containing nutrient media comprising hybridomas that secrete antibody molecules with appropriate antigen specificity. This culture is maintained under conditions and for a time sufficient for hybridomas to secrete antibody molecules into the medium. The antibody containing medium is then collected. Well known techniques can then be used to further isolate antibody molecules.

Useful media for the preparation of these compositions are well known in the art and are commercially available, including synthetic culture media, inbred mice, and the like. Examples of synthetic media include Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8: 396 (1959)) supplemented with 4.5 g / l glucose, 20 mM glutamine, 20% calf serum. An example of an inbred mouse system is Balb / c.

Methods of making monoclonal antibodies are also well known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983). Typically membrane surface proteins or peptide analogues, either alone or conjugated to an immunogenic carrier, are used as immunogens in the methods described above for the preparation of monoclonal antibodies. Hybridomas are screened for their ability to produce antibodies that immunoreact with membrane surface proteins.

The invention also provides therapeutic compositions useful for carrying out the methods of treatment of the invention. Such therapeutic compositions contain a pharmaceutically acceptable excipient (carrier) and one or more membrane surface proteins as described above (ie membrane surface proteins of cells, bacteria or viruses), in particular sperm antigen polypeptide analogs or fragments thereof as active ingredients. Include in form. In a preferred embodiment, the composition contains sperm antigens or mixtures thereof that can inhibit pregnancy.

The preparation of therapeutic compositions containing a polypeptide, analog or active fragment as active ingredient is well known in the art. Typically such compositions are prepared as injectables, such as liquid solutions or suspensions, but may also be prepared in solid form suitable for dissolution and suspension in liquid prior to injection. The formulation may be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and can be used in combination with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like and combinations thereof. If desired, the composition may also contain auxiliary substances such as wetting agents and emulsifiers and / or pH buffers in amounts less than other ingredients, which enhance the efficacy of the active ingredient.

Polypeptides, analogs or active fragments may be formulated into therapeutic compositions in the form of neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid and mandelic acid (formed with the free amino groups of the polypeptide or antibody molecule). Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. have.

The therapeutic polypeptide, analog or active fragment containing composition is typically administered intravenously, for example as a unit dose of injection. The term "unit dose" as used in connection with the therapeutic composition of the present invention refers to physically discrete units suitable as unit doses to humans, each unit having a predetermined amount of active substance calculated to produce the required therapeutic effect and Diluents (ie carriers or vehicles) are contained together.

The composition is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount to be administered depends on the patient to be treated, the immune system dose of the patient to use the active ingredient, and the degree of binding dose required. The exact amount of active ingredient to be administered will be at the discretion of the physician and will vary from individual to individual. However, the range of suitable dosages is from about 0.1 to 20, preferably from about 0.5 to about 10, more preferably from 1 to several mg of active ingredient per kg of body weight per day per day and will vary depending on the route of administration. Curing regimens suitable for initial administration and second inoculation vary, but typically, after initial administration, repeated doses are administered at different intervals of one hour or more, followed by another injection or other administration. Alternatively, continuous intravenous infusion is considered sufficient to maintain a blood concentration of 10 nM to 10 μM.

Therapeutic compositions may also include effective amounts of one or more antagonists or antibodies to membrane surface proteins, and active ingredients such as antibiotics, steroids, and the like.

Another aspect of the invention is the expression of a protein encoded by a DNA sequence obtained through labeling, analysis and sequencing. As is well known in the art, DNA sequences can be expressed by operably linking them to expression control sequences in appropriate expression vectors, which are used to transform the appropriate single cell host.

The meaning that the DNA sequence of the present invention is operably linked to an expression control sequence is that the start codon ATG, even if not originally part of the DNA sequence, exists in the correct reading frame upstream of the DNA sequence.

Various host / expression vector combinations can be used to express the DNA sequences of the invention. Useful vectors can consist, for example, of chromosomal, non-chromosomal and fragments of synthetic DNA sequences. Suitable vectors include derivatives of SV40, known bacterial plasmids (e.g., E. coli plasmid col E1, pCR1, pBR322, pMB9 and derivatives thereof), plasmids such as RP4, phage DNA, e.g. phage λ such as NM989. Numerous derivatives and other phage DNA (eg M13 and filament single stranded phage DNA), yeast plasmids (eg 2 μ plasmid or derivatives thereof), vectors useful in eukaryotic cells (eg useful in insect and mammalian cells) Vectors), vectors derived from combinations of plasmids and phage DNA (eg, plasmids modified using phage DNA or other expression control sequences), and the like.

Any of a variety of expression control sequences (sequences that control the expression of DNA sequences operably linked to them) can be used to express the DNA sequences of the invention in these vectors. Such useful expression control sequences include, for example, early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, lac system, trp system, TAC system, TRC system, LTR system, major operators and promoters of phage λ. Region, regulatory region of the fd coout protein, promoter of 3-phosphoglycerate kinase or other glycolysis enzymes, promoter of acid phosphatase (eg Pho5), promoter of yeast α-mating factor, and In addition, there are sequences known to control gene expression of prokaryotic or eukaryotic cells or viruses, and various combinations thereof.

Various single cell hosts can be used to express the DNA sequences of the invention. These hosts are well known eukaryotic and prokaryotic hosts, for example fungal and animal cells, such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces strains, yeasts, for example CHO, R1.1, BW and LM cells, African savannah monkeys. Cells (eg, COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (eg SF9), and tissue cultured human cells and plant cells.

In expressing the DNA sequences of the invention, it will be understood that not all vectors, expression control sequences and hosts exhibit equally good functions. In addition, not all hosts function identically with the same expression system. However, those skilled in the art will be able to select appropriate vectors, expression control sequences and hosts without undue experimentation to achieve the desired expression without departing from the scope of the present invention. For example, the host must be considered in selecting the vector since the vector must function in the host. The number of copies of the vector, the ability to modulate the number of copies, and the expression of any other protein (eg antibiotic marker) encoded by the vector will also be considered.

Several factors will typically be considered in selecting an expression control sequence. Such elements include, for example, the compatibility with the specific DNA sequence or gene to be expressed, with respect to the relative strength of the system, its regulatory capacity, and in particular its potential secondary structure. Suitable single cell hosts are for example compatible with the host of the product encoded by the DNA sequence to be expressed as well as the compatibility of these with the selected vector, their secretion characteristics, their ability to accurately fold the protein, and their fermentation requirements. The toxicity and ease of purification of the expression product will be considered in consideration.

In view of these and other factors, one skilled in the art can construct a variety of vector / expression control sequences / host combinations that express the protein encoded by the DNA sequences of the present invention either in fermentation or in large animal cultures.

The cDNA is preferably cloned under the control of the bacteriophage T7 RNA polymer / promoter system in E. coli expression vector pET22b (+) (Novagen, Madison, WI). If an endogenous signal peptide is present, the signal peptide can be replaced by the bacterial signal pelB sequence. To be expressed in the pET vector, the foreign gene is (a) pelB sequence at the 5 'end (which facilitates the release of the generated protein into the periplasmic space) and (b) 6 histidine stretches at the 3' end. In-frame fusion is essential with (his-tag) (functioning as a anchor for purification by fixed ion affinity chromatography). Polymerase chain reaction (PCR) cloning methods can be used to make constructs for gene expression, allowing full-length proteins to be expressed whenever possible. Inserts can be verified through sequencing. Clones bearing the coding region of the antigen can be grown in 3X harsh cultures containing ampicillin (100 μg / ml) at 37 ° C. Once the cells reach A ₆₀₀ = 0.6, 0.4 mM IPTG can be added to the culture to induce expression. The negative control host cell culture can be treated in the same manner.

To optimize the recovery of the expressed protein, the construct can be analyzed over time for expression induction in E. coli. For large scale production of recombinant constructs, 14 liter cultures can be grown in New Brunswick ML410 fermentors. The six histidine residues at the carboxy terminus facilitate the process purification process using His-Bind® metal chelating resins. To demonstrate the final purity of the isolated recombinant antigen, Coomassie blue [amido black] and silver staining can be performed on SDS-PAGE gels loaded with varying concentrations of purified product.

Affinity purified antibodies can be generated against recombinant proteins and tested on two-dimensional immunoblot to determine whether the same protein spots originally selected were immunoreactive, thereby obtaining additional [immunological identity] evidence To secure. Role for given proteins in functional responses mediated at the cell or viral surface by studying whether these antibodies bind to sperm surfaces by immunofluorescence and FACS and assessing their impact on protein function in various in vitro assays can confirm.

It should also be borne in mind that membrane surface protein analogs can be prepared from the nucleotide sequence of a protein derived within the scope of the present invention. Analogues such as fragments can be prepared, for example, by pepsin digestion or recombinantly produced material isolated by gel analysis. Other analogues, such as mutant proteins, are made by standard positional mutagenesis of the coding sequence.

As mentioned above, DNA sequences encoding membrane surface proteins can be produced synthetically rather than cloning. DNA sequences can be designed with appropriate codons for amino acid sequences. When the sequence is used for expression, a codon that is usually preferred for the host in mind will be selected. Complete sequences can be prepared by standard methods and assembled from overlapping oligonucleotides into complete coding sequences. See, eg, Edge, Nature, 292: 756 (1981), Nambair et al., Science, 223: 1299 (1984), Jay et al., J. Biol. Chem., 259: 6311 (1984). See.

Synthetic DNA sequences allow convenient construction of genes expressing membrane surface protein analogs or “mutant proteins”. Alternatively, the DNA encoding the mutant protein can be made by localized mutagenesis of the native gene or cDNA, and the mutant protein can be made directly using conventional polypeptide synthesis.

The present invention extends to methods of making antisense oligonucleotides and ribozymes that can be used to interfere with expression of membrane surface proteins at the translational level. In this approach, antisense nucleic acids and ribozymes are used to block specific mRNAs with antisense nucleic acids or cleavage to ribozymes to disrupt translation of the mRNA.

Antisense nucleic acids are DNA or RNA molecules complementary to at least a portion of specific mRNA molecules (Weinstraub, 1990; Marcus-Sekura, 1988). Antisense nucleic acids hybridize to mRNA in cells to form double stranded molecules. The cell does not translate this double stranded mRNA. Thus, antisense nucleic acids prevent the mRNA from being expressed as a protein. Oligomers and molecules of about 15 nucleotides that hybridize to AUG initiation codons are particularly effective because they are easy to synthesize and will have little problem compared to large molecules when introduced into cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988, Hambor et al., 1988).

Ribozymes are RNA molecules that have the ability to specifically cleave other single stranded RNA molecules in a manner somewhat similar to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs are capable of cleaving their introns. The researchers could modify the nucleotide sequences of these RNAs to obtain molecules that recognize and cleave specific nucleotide sequences within certain RNA molecules (Cech 1988). Since ribozymes are sequence specific, only mRNAs with specific sequences can be inactivated.

The researchers identified two types of ribozymes, the Tetramhymena type and the "hammerhead" type (Hasselhoff and Gerlach, 1988). Tetrahimena-type ribozymes recognize four base sequences, while the "hammerhead" type recognizes 11-18 base sequences. The longer the recognition sequence, the more likely it is to be recognized only within the target mRNA species. Thus, hemmerhead ribozymes are more preferred for inactivating specific mRNA species than tetrahimena ribozymes, and 18 base recognition sequences are preferred over shorter recognition sequences.

Using the DNA sequences described herein, antisense molecules to mRNA molecules of membrane surface proteins and their ligands and ribozymes that cleave this mRNA can be prepared.

The invention also provides a method for detecting the presence of specific cells or infectious mediators or antibodies thereto (ie, in the serum of a patient) based on their ability to bind or compete with binding of membrane surface proteins. And various diagnostic uses. As mentioned above, membrane surface proteins can be used to prepare antibodies against them in a number of known ways, and such antibodies can then be isolated and used for testing to determine the presence of specific target cells or infectious mediators. There will be. Such antibodies are used to analyze cell surface proteins, especially for sperm proteins such as sperm aggregation, sperm immobilization, surface immunofluorescence, FACS analysis, sperm invasion analysis, translucent band analysis, positioning on two-dimensional surface maps. Can be used.

As detailed above, the antibody (s) can be prepared and isolated by standard methods, including well known hybridoma techniques. For the sake of simplicity, herein the antibody (s) to the membrane surface protein will be named Ab ₁ and antibodies from other species will be named Ab ₂ .

The presence of cells and infectious mediators, including repertoires of membrane surface proteins, can be confirmed by conventional immunological methods applicable to such identification. Many useful methods are known. Three such methods that are particularly useful utilize one of the surface proteins labeled also detectable label, antibody Ab ₁ labeled with the detectable label, antibody Ab ₂ labeled with the detectable label. This method is summarized by the following equation. Where an asterisk (*) denotes labeled particles and “MSP” denotes a membrane surface protein.

A. MSP ^* + Ab ₁ = MSP ^* Ab ₁

B. MSP + Ab ^* = MSPAb ₁ ^*

C. MSP + Ab ₁ + Ab ₂ ^* = MSPAb ₁ Ab ₂ ^*

Since the methods and their applications are all familiar to those skilled in the art, they can be used within the scope of the present invention. Method A, a “competitive” method, is described in US Pat. Nos. 3,654,090 and 3,850,752. Method C is a "switch" method, described in US Patents RE31,006 and 4,016,043. Other methods are also known as the "dual antibody" or "DASP" method.

In each case, the membrane surface protein forms a complex with one or more antibody (s) or binding partners, belonging to this complex is labeled with a detectable label. The fact that the complex is formed and, if necessary, the amount thereof, can be confirmed by known methods applicable to label detection.

From just above the Ab ₂ will be seen that it is a characteristic property of Ab ₂ to react with Ab _1. This is because Ab ₁ generated in one mammalian species was used as an antigen in another species to induce antibody Ab ₂ . For example, Ab ₂ can be induced in goats using rabbit antibodies as antigens. Ab ₂ will thus be an anti-rabbit antibody derived from goats. In this specification and claims Ab ₁ will be referred to as primary or anti-membrane surface protein antibodies, and Ab ₂ will be referred to as secondary or anti Ab ₁ antibodies.

The most commonly used labels in this study are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others.

Many fluorescent materials are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramin, Texas red, AMCA blue, and lucifer yellow. Particular detection agents are anti-rabbit antibodies made in chlorine and conjugated with fluorescein via isothiocyanate.

Membrane surface proteins or their binding partner (s) can be labeled with radioactive elements or enzymes. Radiolabels can be detected by commercially available counting methods. Preferred isotopes may be selected from ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re.

Enzyme labels are likewise useful and can be detected using any of the colorimeters, spectrometers, fluorometers, ammeters, or gas metering techniques currently used. The enzyme is conjugated to the selected particles by reacting with a crosslinking molecule such as carbodiimide, diisocyanate, glutaldehyde and the like. Many enzymes that can be used in these methods are known and can be used. Preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase + peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are cited as other labeling materials and methods as examples for publication.

In another embodiment of the present invention, a commercial test kit suitable for use by a medical professional can be prepared to determine the presence of a membrane surface protein repertoire on a suspect target cell. In accordance with the test techniques as described above, one kind of such kit is at least a labeled membrane surface protein (s) or a binding partner thereof (e.g., an antibody specific for the membrane surface protein) and "competition", "sandwich", Instructions according to the selected method, such as "DASP", are included. The kit may also include peripheral reagents such as buffers, stabilizers, and the like.

Thus, the test kit

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by direct or indirect attachment of a detectable label to a corresponding membrane surface protein or binding partner specific for it,

(b) other reagents and

(c) can be prepared to demonstrate the presence or performance of the cell in the presence of the cell or infectious medium, including instructions for use of the kit.

More specifically, the diagnostic test kit

(a) Generally, the membrane surface protein (or binding partner) as described above bound to a solid phase that forms an immunosorbent, or to an appropriate tag or a number of such end products or the like (or binding partners thereof) Knowing,

(b) other reagents, if necessary;

(c) may include instructions for using the kit.

In other variations, the test kit may be manufactured and used for the above purposes in which it operates according to a predetermined protocol (eg, “competition”, “sandwich”, “double antibody”, etc.),

(a) a labeled component obtained by coupling a membrane surface protein to a detectable label,

(b) one or more additional immunochemical reagents, at least one of which is a ligand or an immobilized ligand, the ligand

(i) a ligand capable of binding to the labeled component (a),

(ii) a ligand capable of binding to the binding partner of the labeled component (a),

(iii) a ligand capable of binding one or more of the component (s) to be identified, and

(iv) selected from the group consisting of ligands capable of binding one or more binding partners of one or more of the component (s) to be identified; and

(c) instructions for carrying out a protocol to detect and / or confirm one or more components of an immunochemical response between the membrane surface protein and its specific binding partner.

According to the above, assay systems can be prepared for screening for potential drugs effective in modulating the activity or binding of membrane surface proteins. The membrane surface protein is introduced into the test system, the prospective drug is introduced into the resulting cell culture, and the culture is examined to determine the amount of addition of the known membrane surface protein or by addition of this potential drug alone. Any change in the binding activity of the cells by the effect was observed. Suitable assays include sperm motility assays or in vitro fertilization. In addition, suitable animal models, particularly animal models for fertilization attempts, include mice and macaques.

Membrane surface proteins can also be used as vaccines and sperm antigens can be used as contraceptives.

Once a number of unique, testicular specific sperm surface immunogens have been identified, they can be naturally and effectively transferred to the contraceptive development pathway through the triage system. Candidate molecules must meet stringent conditions of surface localization, testicular specificity, and activity in at least one functional test. The cDNA cloned and sequenced proteins are tested for tissue specificity. If testicular specific, they will be expressed as recombinant proteins and antibodies to these recombinant proteins will be generated. These antibodies will be used to demonstrate the biologic effects of immunogenic epitopes in surface localization and function tests.

Proteins that have been repeatedly observed by directed labeling methods exposed on human sperm surface proteins include infertile serum, autoantigens defined by sera obtained from vasectomy men, and antigens that cause sperm aggregation. These proteins are candidates for immunocontraception.

In order to avoid autoimmune complications, it is particularly important that the molecules selected for the development of immuno contraceptives exhibit tissue specificity, that is, they are expressed on sperm in the testes, which are their intended target tissues. In toxicity and teratological safety studies, it is better to introduce tissue specificity at the vaccinogen discovery stage than at later stages. This method minimizes unnecessary waste of time and minimizes the resources for immunogens that are widely distributed in tissues and can potentially cause immune conditions. Potential complications of immunization with contraceptive vaccines include 1) immediate hypersensitivity and anaphylactic reactions, 2) delayed hypersensitivity reactions, and 3) autoimmune reactions by immune responses to endogenous antigens of immunized patients and autoimmune diseases. . Many of these concerns can be avoided by selecting nonreactive immunogens specific for testes and sperm. Immunohistochemical, Northern and RT-PCR studies on the tissue specificity of the hypothetical contraceptive immunogen provide important evidence. Northern blot and RT-PCR methods provide evidence of tissue specificity at the mRNA level, but cannot detect epitopes in selected vaccinogens that may be present in other unrelated molecules (structural folding or other types of molecular imitation). because of). Immunohistochemical testing thus complements molecular methods and may reveal unpredictable cross-reactive epitopes based solely on comparisons of primary amino acid sequences.

Northern blots, RT-PCR and immunohistochemical methods can be combined to identify spouse specific antigens that are at less risk for autoimmune diseases. This provides information on the stage of spermatogenesis where the presented protein is first expressed, which means that the testicular specific gene is active before or after meiosis. This knowledge is intimately related to identifying posterior meiosis genes for studying transcriptional regulation, means for regulating spermatogenesis, and possible pathways for finding means for developing male contraception.

To advance immunogenicity and modification attempts of candidate sperm immunogens, their homologues can be cloned in mice and monkeys and the recombinant proteins expressed and purified.

Homologs of both monkeys and mice can be cloned using testicular libraries as reported in the prior art [Proc. Natl. Acad. Sci. USA 84: 5311-5315 (1984); Mol. Repd. Dev. 34: 140-148 (1993)]

Female B6AF1 mice 6-8 weeks of age can be immunized with about 20 μg of homologous recombinant immunogen. Immunogens can be emulsified with complete blood Freund's adjuvant (CFA) in the Eastern volume for a final volume of 100 μl. Animals in the control group will be administered PBS / CFA. Two injections can be made at the base of each animal's tail. Two or three reimmunizations of incomplete Freund's adjuvant may be given at two week intervals. Blood was collected by tail bleeding prior to immunization, 2 weeks after immunization and 7-10 days after each reimmunization. Serum was analyzed by ELISA for antibodies using sperm extract and recombinant immunogen as targets. Serum was further tested for responsiveness to natural proteins using immunofluorescence and immunoblot analysis of mouse sperm and sperm extract, respectively. Starting one week after the last reimmunization with recombinant immunogen, each female mouse was kept in fertile male mice and cages. The female should be checked each morning to see if there is a vagina that indicates mating. On the 15th day after the males were introduced into the cages, the females were killed to count fetuses.

Macaques were injected intramuscularly with 500 μg of squalene-arlacel A and then re-immunized with 200 μg or control squalene every 3 weeks. Serum, cervical mucus and fallopian fluid were collected at weekly intervals prior to immunization (collected through surgically implanted fallopian cannula). Antibodies can be measured by ELISA against both recombinant immunogens and natural sperm extraction targets. Serum can be further tested by immunoblot and immunofluorescence assays using monkey and human sperm and sperm extracts for responsiveness to natural proteins. Serum can then be further tested for their ability to present sperm function tests. In addition, recombinant immunogens that produce high titer concentrations of antibodies that cross react with the sperm surface and block one or more functional tests will undergo fertilization attempts.

The macaques are immunized as above with recombinant immunogen (15 animals) or control squalene (15 animals) for fertilization attempts. Serum is collected at weekly intervals before immunization. Antibodies are evaluated by ELISA, Western and immunofluorescence assays. After the final immunization, each female is kept in the cage simultaneously for 5 days with fertile male during fertilization during the cycle beginning 3 days prior to ovulation. Mating begins during the third cycle after initial immunization and continues for 9 consecutive weeks or until female pregnancy is confirmed. Pregnancy is terminated by administering sulfostone, an anti-luteinizing hormone. Observe daily to detect abortion 6 weeks before pregnancy.

In fertilization attempts, the implications of testing between fertilization rates of vaccinated and control animals may depend primarily on nonparametric randomization procedures. Fisher's rigorous testing and tests based on binomial ratios can be used. In addition, the significance of differences between vaccinated and control groups in fertilized and uncensored animals can be confirmed by traditional Chi square analysis. Time to pregnancy was analyzed using several life table methods, including the nonparametric comparison of survival curves, the Mantel-Haenszel test, several possible models, and a proportional risk regression model. Similar procedures can be used to investigate the relationship between antibody titer concentrations and time to fertilization results. For example, studies may provide guidance for comparing antisperm antibody levels of fertilized animals vaccinated with unfertilized animals vaccinated.

In addition, the amounts of antisperm antibodies of animals fertilized at different times were compared. For group reactions that produce continuous results, two sample comparisons can be performed using the nonparametric Wilcox rank sum test and the conventional two-sample t-test. In some cases, regression models (including variance and covariance analysis) can be tested with valuation and transformation of data to optimize statistical evaluation and inference and improve model suitability.

If immunity in mice or monkeys causes high infertility, the results indicate that this immunogen needs to be studied continuously. If high efficacy appears in primates, human immunogens will be useful as agents for human immunogenicity testing (Phase 1). If the effect on fertility is moderate, this immunogen can be included as a component of a multi-determinant contraceptive vaccine formulation. If a given immunogen does not show any effect, this result should be (1) excluded for further study or (2) retested with another carrier system that can lead to higher or more sustained titer concentrations in the female reproductive tract. .

If 75% or more infertility is obtained for a given immunogen in the test group, two possible options are to (1) continue mating infertile animals to begin reversibility / fertility persistence studies or (2) perform histopathology. If the latter is chosen, tissue can be obtained as a result of fertilization attempts from vaccinated females and soaked in fixative. Standard formaldehyde fixation, paraffin impregnation and H & E staining were performed on the cerebellum, cerebral (frontal and temporal regions), brain stem, myocardium, artery, skeletal muscle, interest, spleen, letter, adrenaline, stomach, gallbladder, The duodenum, jejunum, colon, kidney, bladder, ovary, uterus, mammary gland, umbilical cord, and parotid gland can be performed.

Effective contraceptive vaccines against sperm antigens require an immune response that results in maximum antibody titer concentrations in the female reproductive tract. Systemic immunity with sperm antigen SP-10 triggers an IgG antibody response in the fallopian fluid, and cervical immunization provides another effective immunization pathway. If systemic immunity alone produces an insufficient amount of antisperm antibody in the gonad fluid in an immunogenic test, the immunization system including cervical immunization selectively increases the amount of IgA and IgG antibodies in the female macaque gonad fluid. Can be tested.

Although the present invention has been described in general, it is to be understood in greater detail with reference to specific specific embodiments, but the embodiments are provided herein by way of example only, and unless otherwise stated, the scope of the present invention is not limited thereto.

Experimental procedure

material

Ammonium persulfate, BSA, citric acid, PMSF (phenylmethylsulfonyl fluoride diethanolamine, glycerol, urea (Sigma Ultra), Trizma Base (Sigma Ultra), Nonidet P40, CHAPS, PDA (N, N'-Diacrylo Ilpiperazine), DTT, iodoacetamide, leupeptin and pepstatin A were obtained from Sigma Inc. EDTA was purchased from JT Baker Silver nitrate was obtained from Mallinckrodt Chemicals Sodium bicarbonate, sodium chloride and sodium hydroxide (all ACS Grades) were purchased from Fisher, SDS (Sodium Dodecyl Sulfate, Ultrapure), Glycine (Electrophoretic Grade) and Sodium Phosphate were obtained from ICN Percoll, Ampholine pH 3.5-5, pH 5-7, pH 7- Carbamylation measurement kits for 9 and pH 3.5-10, Pharmalyte pH 6.5-9, pH 5-8 and pH 8-10.5 and two-dimensional electrophoresis were purchased from Pharmacia Biotech. ECL) kit is Amersham Corp. (for detection of peroxidase conjugates) And Tropix (detection of alkaline phosphatase conjugates) Protogold for staining of blotted proteins was purchased from Goldmark TLCK (Np-tosyl-L-lysine chloromethyl ketone) and TEMED from Boehringer Mannheim. Lectin conjugates were obtained from Vector Na ¹²⁵ I without carrier was purchased from Amersham Corp. Sensitivity Enhancement Screens NEF-490 and NEF-491 were from Du Pont HRP conjugated avidin and two-dimensional SDS-PAGE standards NHS-LC-biotin, iodine-bead and AP-conjugated avidin were purchased from Pierce All secondary antibodies used in Western blotting were purchased from Jackson ImmunoResearch Lab.

<Example 1>

Sperm Preparation

Masturbation was obtained from a normal healthy young male through masturbation. Only ejaculates with normal semen parameters (28) were used in this study. Individual semen samples are liquefied at room temperature (normally for 1/2 to 3 hours) and mature sperm are separated from seminal plasma, immature germ cells, and nonsperm cells (primarily leukocytes and epithelial cells) by Percoll density gradient centrifugation. I did it. The semen, which became liquid, was carefully added dropwise to a two-layer Percoll density gradient consisting of 80% (1 ml lower layer) and 55% (2 ml upper layer) Percoll isotonic solution prepared with Ham's F-10 medium. After centrifugation at 300 x g for 18 minutes at room temperature, sperm pellets were collected at the bottom of the 80% layer and washed three times with Ham's F-10 medium by centrifugation at 450 x g. The cells were counted before the last centrifugation and the pellets used for directional labeling. Using an optical microscope, it was confirmed that abundant dynamic sperm was abundant, and the motility of all samples was> 90%.

<Example 2>

Semen Plasma Analysis

Semen stromal samples from two patients who underwent vasectomy were analyzed by two-dimensional electrophoresis. After collecting the sample, it was liquefied at room temperature and examined under a microscope to confirm that there were no sperm or immature germ cells, and centrifuged at 10,000 xg for 5 minutes to remove the reproductive cells and prostate crystals, -70 ℃ Divided into aliquots and stored until use.

<Example 3>

Percoll purified sperm were suspended in Ham's F-10 medium to a final concentration of 20 × 10 ⁶ / ml. Washed iodine-beads (one bead per 8 × 10 ⁶ sperm) and carrierless Na ¹²⁵ I (10 μCi per 10 ⁶ sperm) were added to the sample. Radioiodination was performed by incubating this sample on a shake table at 20 ° C. for 10 minutes. The cells were removed from the iodine-bead using a pipette, immediately subjected to a second Percoll density gradient centrifugation, and then washed three times with Ham's f-10 medium. The cells were counted before the last wash and the resulting pellets were used for extraction of sperm protein (see below). Magnetic radiographs were performed using component sandwiches in the order of sensitivity enhanced screens, blots, bilayer films and sensitivity enhanced screens. X-ray films were usually exposed for three weeks.

<Example 4>

Biotinylation

Percoll purified sperm were suspended in Dulbecoo phosphate buffered saline containing 3 mg / ml of NHS-LC-biotin (about 5 mM) to a final concentration of 50 × 10 ⁶ sperm per ml. Biotinylation of the sperm surface was performed by incubating the sample on a shake table at 37 ° C. for 10 minutes. Sperm was then centrifuged in a second Percoll density gradient and washed three times with Ham's F-10 medium. Cells were counted before the last wash and the resulting pellets used for solubilization or stored at -70 ° C.

After a preliminary study to optimize the detection of biotinylated protein, biotinylated sperm protein was detected after electrophoresis to NC membrane as follows. The two-dimensional western blot was washed twice with PBS (pH 7.4) and then incubated in PBS containing 5% gelatin and 0.1% Tween 20 for 1 hour at 20 ° C. to block excess binding sites on the nitrocellulose membrane (29 ). It was then washed with PBS for 5 minutes and incubated with alkaline phosphatase conjugated avidin (50 μl in 250 ml PBS containing 0.5% gelatin and 0.01% Tween 20) at 20 ° C. for 1 hour. Chemiluminescence detection of alkaline phosphatase and substrate CSPD was performed according to the manufacturer's instructions (Tropix). Colorimetric detection of AP was performed as described above using the substrates BCIP and NBT (17). Samples from five donors (including samples from two of four donors tested by radioactive iodide) were examined by vectorially labeling techniques.

<Example 5>

Solubilization Method

Sperm Lysis Buffer (A) (2% (v / v) NP-40, 9.8 M urea, 100 mM DTT, 2% (v / v) Ampoline pH 3.5-10, Protease Inhibitor 2 mM PMSF, 5 mM Io Solubilized in conventional manner with doacetamide, 5 mM EDTA, 3 mg / ml TLCK, containing 1.46 μM pepstatin A and 2.1 μM leupeptin. 5 × 10 ⁸ cells per ml were solubilized by constant shaking at 4 ° C. for 60 minutes. Insoluble material was removed by centrifugation at 10,000 xg for 2 minutes and the supernatant was subjected to the first electrophoresis dimension. Frozen semen plasma samples from patients undergoing vasectomy were thawed by adding 4 volumes of Lysis Buffer A containing the protease inhibitor described above.

In another experiment anionic / amphoteric lysis buffer consisting of 2% (w / v) SDS, 3% (w / v) CHAPS, 7.2 M urea, 100 mM DTT, 4% ampoline pH 3.5-10 and a protease inhibitor ( B) (provided by Hochstrassser (30)). Solubilized at a sperm concentration of 3.5 × 10 ⁸ cells per ml at 22 ° C. for 45 min. Samples were agitated every 5 minutes while inverting the microcentrifuge tube to minimize DNA unfolding. This is because constant shaking or heating in the presence of SDS and reducing agent results in solubilization of nuclear envelope and non-folding of sucoil DNA (Naaby-Hansen and Bjerrum, closed results). Protein concentrations were determined using the Pierce biosuccinic acid method according to the manufacturer's specifications and using bovine serum albumin (BSA) as standard.

<Example 6>

Electrophoresis

Isoelectric focusing (IEF) was performed in 15 x 0.15 cm acrylamide rods using the gel compositions suggested in Hochstrasser et al. (30) or Cells et al. (16). The carrier ampoline composition may be 20% pH 5-7, 20% pH 7-9 and 60% pH 3.5-10, or 28% pH 3.5-5, 20% pH 5-7, 7% pH 7-9 and 45% pH was 3.5-10. For each rod, 65 μl of sperm extract (approximately 0.15 mg of protein) or 35 μl of seminal plasma sample (approximately 0.15 mg of protein) were added. The samples were slowly applied to the tubes by filling the samples with buffer containing 5% NP-40, 1% ampoline pH 3.5-10, 8 M urea and 100 mM DTT. Focusing was performed for a total of 19,300 volt-hours using voltage steps of 2 hours at 200 V, 5 hours at 500 V, 4 hours at 800 V, 6 hours at 1200 V, and 3 hours at 2000 V.

Non-equilibrium pH gradient electrophoresis (NEPHGE) was performed using a gel composition as described in Celis et al. (16) in a 14 x 0.15 cm acrylamide rod. The carrier amphoteric composition was 37% pH 5-8, 13% pH 6.5-9, 37% pH 8-10.5 and 13% pH 3.5-10. For each rod, 55 μl of sperm extract (approximately 0.13 mg of protein) or 30 μl of seminal plasma sample (approximately 0.13 mg of protein) were applied. These protein concentrations gave a similar spot size on the NEPHGE gel as obtained by slightly higher concentrations on the IEF gel using Hochstrasser's protocol. Electrophoresis was performed for a total of 9800 volt-hours (11 hours at 400 volts and 9 hours at 600 volts).

In a 0.15 cm thick, 16 x 16 cm or 16 x 20 cm slab gel, using a linear gradient gel (T = 7.5-15% or 9-15%) in a Protean II xi Multi-Cell instrument (Bio-Rad) 2D SDS-PAGE was performed at. Silver staining was performed according to Hochstrasser et al. Electrophoresis to the nitrocellulose membrane was carried out (23) as described above. Electrophoresis to the PVDF membrane (0.2 μm pore size, Pierce) was performed using Matsudaira (32) (10 mM 3- [cyclohexylamino] -1-propanesulfonic acid, 10% methanol, pH 11) as described in Herizel et al. (31). It was carried out using a moving buffer composition. In a conventional manner, six two-dimensional gels were simultaneously moved using three transblot cells (Bio-Rad) connected to the same power source for two hours at constant current intensity (0.5 A).

<Example 7>

To perform electrophoretic transfer of 2-D separated polypeptides, the immobilized membranes were washed twice for 5 minutes at PBS pH 7.4 and incubated for 30 minutes at room temperature in PBS pH 7.4 containing 5% milk powder for excess The binding site was blocked and 0.05% Tween 20. 2-D blots were incubated with 100 ml of primary antibody at 22 ° C. for 2 hours or overnight at 4 ° C. with constant slow shaking. The antibodies used were as follows. Antiactin (mouse mAb clone C4, from Boehringer Mannheim, 0.3 μg / ml), anti-β-tubulin (mouse mAb Tu 27BC, hybridoma supernatant obtained from Dr. Robert Bloodgood, University of Virginia) Diluted to 5)), anti-α-tubulin (mouse mAb, clone DM-1A, manufactured by Sigma, diluted 1: 10,000), anti-SP-10 (mouse mAb MHS-10, diluted 1: 5,000) ), Anti-fibrous sheath protein (mouse mAb S69, obtained from University of Vancouver Dr. CGLee, diluted 1: 1,000) and anti-PH-20 (rabbit polyclonal Ab R-10, UC Obtained from Dr. Paul Primakoff of Davis, diluted 1: 3,000). Enzyme bound secondary antibody enzymes (goat anti mouse Ig and goat anti rabbit Ig) were diluted 1: 5000 in PBS + 0.05% Tween 20 and blots were incubated at 20 ° C. for 1.5 hours. None of the secondary antibodies bound to the bloated human sperm protein. Horseradish peroxidase binding was visualized by calorimetry using DAB as substrate or by ECL (Amersham Corp.) using the manufacturer's protocol.

To detect phosphorylated tyrosine residues, mAb RC-20 (manufactured by Transduction Laboratories) was used. Blots were blocked by incubation at 1 ° C. in 1% BSA, 0.1M NaCl, 0.1% Tween 20 for 20 min at 7 ° C. for 20 min. MAb RC-20 bound with horseradish peroxidase was diluted to about 1: 2500 in this buffer and blots were incubated at 37 ° C. for 20 minutes and binding was detected by ECL.

In order to confirm the exact location of the immunoreactive antigen in the complex 2-D protein pattern, in several experiments the antibody culture was first stained with Protogold (Goldmark Biologicals). The manufacturer's protocol was followed by a modified method where the membrane blocking was done in 0.05% Tween 20 for 30 minutes. The concentration of Tween 20 used in place of the recommended 0.3% Tweem 20 resulted in better spot separation with a less dense staining pattern in the high density polypeptide region.

<Example 8>

Gel Staining and Computer Analysis

Stained gels were screened wet using a high resolution Kodak camera and the information was digitized on a SUN computer. X-ray films obtained from radiographs and chemiluminescent experiments were screened with either Kodak cameras or Howtek scanmaster 3+ laser scanners. The 2-D image obtained from this was analyzed by Bioimage program "2D analyzer". Sperm samples from 40 donors were analyzed on a 500 2-D gel to optimize the procedure. Sperm samples from eight donors were established on a 2-D gel to establish a reliable protocol and protein patterns were compared to reduce the likelihood of sorting into artificial spots. Spots from two or more analyzes from each donor were significant. As many reference spots as possible were manually matched using an actuator followed by a computer program of spot matching on different gel images (up to 100 allowed by this software for silver stained gels). Isoelectric points and molecular weights of unknown spots were inserted manually into the computer program based on the co-moving internal standard, and the molecular weight results were also verified manually by calculating from the inverse plot. Internal standards (BIO-RAD) are hen eggs white cone albumin type I (MW 76,000, pi 6.0, 6.3 and 6.6), bovine serum albumin (MW 66,200, pi 5.4, 5.5 and 5.6), beef muscle actin (MW 43,000, pi 5.0 and 5.1), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MW 36,000, pi 8.3 and 8.5), bovine carbonate dehydrogenase (MW, 31,000, pi 5.9 and 6.0), soybean trypsin inhibitor (MW 21,500, pi 4.45) and myoglobin in horses (MW 17,500, pi 7.0). Carbamylated creatine phosphokinase (MW 40,000) and apparent pi range 4.9-7.1) and carbamylated GAPDH (MW 36,000 and apparent pi range 4.7-8.3) from Pharmacia were used as pi markers.

result

Silver stained 2-D gels (20 x 16 cm) of human sperm protein solubilized with nonionic detergent / urea resulted in 1397 different protein spots (963 by IFR / PAGE, 434 by NEPHGE / PAGE) Was separated. 1345 of 1397 proteins were identified in samples of two or more donors. When anionic / zwitterion lysis buffer was used for sperm solubilization, 1191 proteins (838 by IFR / PAGE and 353 by NEPHGE / PAGE) were isolated (A and B in FIG. 9A). Minor deviations were observed between donors, but the protein pattern was quite reproducible. For example, FIG. 2 illustrates the difference in abundance of 39.5 kDa protein among three donors.

The 2-D pattern of human sperm protein is significantly different from the 2-D pattern of human sperm plasma (HSP) protein (FIG. 1). After 2-D electrophoresis separation, more than 300 semen protoplast proteins were visualized by silver staining (C and D in FIG. 1). Semen protoplast polypeptides co-migrating with sperm protein were identified by computer matching silver stained gels containing sperm protein, semen protoplast protein, or a mixture of both, which ensured an accurate comparison. This approach identified nine proteins with MW greater than 20 kDa as sperm coated proteins derived from semen protoplasts (indicated by arrows in FIG. 1). All nine proteins were directionally labeled with radioiodine or biotin, or both (Figures 7, 8 and 11). In general, it is difficult to identify sperm-coated proteins with molecular weights of 20 kDa or less derived from semen protoplasts, as Edwards et al. (33) has observed, particularly in the basic pH range. (D in FIG. 1). In one experiment, a silver stained gel containing sperm collected after 1 hour was compared to a gel loaded with protein from the same sample liquefied for 3 hours. No significant difference between protein patterns could be detected, indicating that major modifications of seminal surface proteins were not induced by sperm protease after liquefaction (not shown).

<Example 9>

Radioiodination of Human Sperm Surface Proteins

The kinetics of incorporation of ¹²⁵ I into human sperm protein with or without an iodine-bead catalyst were investigated. The optimal reaction time was 10 minutes, after which the rate of radioiodine incorporation into the sperm pellets decreased, indicating that it was saturated with surface available phenol (FIG. 3). In the absence of beads coated with a catalytic amount of N-chlorobenzenesulfonamide, ¹²⁵ I was incorporated into the cells at less than 1%. Although radioiodine was incorporated at higher rates with higher concentrations of beads, the use of more beads resulted in gradual breakdown of the membrane, radiolabeling of internal protein standards and decreased sperm motility. We therefore reduced the number of oxidized beads to one bead per 8 × 10 ⁶ sperm. This reduced radioactive iodine incorporation by 50% compared to the former bead / cell ratio, but retained the motility of the posterior sperm.

Radioautomagnography was performed from nitrocellulose or PVDF blots immunostained with antibodies to cytoskeletal and intracellular proteins to determine whether radioiodination occurred only at the sperm surface (FIGS. 4, 5 and 6). ). Sperm populations were separated by Percoll density gradient centrifugation immediately after radioiodization to remove the tip that had been damaged or reacted with the cells from this assay. In the presence and absence of percol density gradient centrifugation [FIG. 4B] and in the absence [A of FIG. 4], when comparing sperm iodide, the extract of sperm subjected to secondary density gradient centrifugation may contain less labeled protein. However, the resolution of each protein is higher, and intracellular control marker proteins (adduct proteins, SP-10, FIGS. 4 and 6; actin, FIGS. 4 and 5; fibrous component, FIG. 9; and tubulin, FIG. 5 )) Appeared to be unlabeled. This strongly supports the surface specificity of the modified radioiodination procedure. Comparing the extracts of radioiodinated sperm with and without reducing agents identified a number of surface proteins involved in the high molecular weight complex stabilized by the interchain disulfide bridge (FIG. 5).

Sperm samples from four donors were analyzed after radioiodation, postlabeled percol density gradient separation, and extraction with NP-40 and urea. The pattern of radioiodinated protein was significantly reproducible from one donor to another as can be recognized through a comparison of two donors (FIG. 7A + B to FIG. 7C + D). Computer image analysis of gels from four donors revealed radioiodination of 181 sperm surface proteins (103 IEF / PAGE and 78 NEPHGE / PAGE) with Mrs ranging from 5 to 150 Kd and pIs ranging from pH 4 to 11. Appeared to be constant. Minor donor interactions were notable at relative concentrations as well as the charge of some labeled proteins, such as the 90-95 Kd protein complex indicated by the arrows in FIG. 7, as measured by optical density. High levels of reproducibility from one experiment to another can be further understood by comparing the iodinated proteins in FIGS. 4 and 7.

<Example 10>

2-D patterns of biotinylated proteins from sperm samples incubated with sulfonated NHS-LC-biotin for 10, 20, 30 and 45 minutes were compared using the ECL detection method (not shown). No differences were observed between multiple labeled proteins after 10-20 minutes of exposure to biotin. However, longer incubation times (30-45 minutes) resulted in progressively labeled cytoskeleton and intracellular proteins, so a 10 minute biotinylation period was used in subsequent studies. Adding 1% bovine serum albumin to the initial wash buffer to react with residual biotin (as recommended in many biotinylation processes) will cause a significant amount of biotinylated BSA to adhere to the sperm surface, so instead of labeling the sperm sample percol density Gradient centrifugation.

8A and 8 show chemiluminescent films of two-dimensional gels containing biotinylated sperm protein solubilized by nonionic lysis buffer. Biotinylated sperm proteins from five donors were separated by IEF and NEPHGE and the images were compared by computer analysis. 228 biotinylated protein spots with a Mr of 5 to 120 Kd and a pH range of 4 to 10 were isolated and sorted.

Interestingly, when the biotinylated sperm protein was incubated with AP-avidin only (C and D in FIG. 8) as a control, a group of five sperm proteins with Mr 76 kDa and pI 6 to 6.5 bound to the label avidin. It was. The properties of this endogenous avidin binding protein are currently being studied. Endogenous alkaline phosphatase activity was not detected in these and other proteins when control blots were developed without preexposure to AP-avidin (not shown).

When anion / Zwitter lysis buffer B was used to extract sperm protein (A and B in FIG. 9A), 208 biotinylated proteins could be isolated (C and D in FIG. 9A). The arrangement of several proteins in the biotin pattern could be recognized from the pattern obtained after extraction with a nonionic detergent (compare C and D in FIG. 9A with A and B in FIG. 8).

To accommodate the specificity of the extraction method for biotinylation and sperm surface, the control cytoplasm was placed immunologically and its biotinylation status was measured relative to ECL-film. None of the internal control actin, tubulin, fibrous component or SP-10 was biotinylated (FIG. 9).

In contrast to the results of known cytoplasmic proteins, the immunoblots of G and H of FIG. 9B were sperm-surface hyalurolilidase PH-20 following SDS / CHAPS extraction and immunoblotting with polyclonal rabbit antiserum R-10. Shows the location of. Two molecular weight forms of PH-20 at 64 and 53 kDa were extracted with SDS / CHAPS (arrows in G and H in FIG. 9B). The 53 kDa antigen was solubilized with NP-40 / urea and then separated into three isoforms (FIG. 8E), solubilized with SDS / CHAPS / urea and five isoforms (G in FIG. 9B). The exact location of the PH-20 antigen in silver staining and biotinylated 2-D gel patterns is shown in Figures A and C of Figure 9a. Biotinylation of the known surface protein PH-20 (34) is used as a positive control for the surface specificity of the labeling procedure.

<Example 11>

Demonstration of surface protein phosphorylated with tyrosine residues

Anti-phosphotyrosine Mab, RC-20 was bound to phosphorylated protein obtained from freshly assessed sperm (FIG. 10). Proteins phosphorylated with the highest amount of tyrosine residues had a molecular weight of 89-95 Kda (arrow in A of FIG. Computer analysis revealed five groups of proteins with phosphorylated tyrosine residues. 22 protein isoforms in this group are marked with asterisks in Table 3.

<Example 12>

Computer Analysis of Two Dimensional Results

11 shows computer generated images of human sperm protein, visualized by NP-40 and UREA after silver staining. Of the 98 proteins labeled by both surface iodide and surface biotinylation, 94 are consistent with silver stained proteins, which are circled in FIG. 11. Mw, pi and relative amounts (based on Gaussian quantitation) are reported in Table III for double labeled surface proteins. Blots of labeled proteins were exposed for various times to obtain optimal detection for both weak and strong signals. To eliminate the possibility that differences in migration between labeled and unlabeled proteins affect the assay, autoradiographs and biotin ECL films were matched to corresponding silver stained gels or gold stained blots. These composite images were then matched to composite images of silver stained gels of unlabeled protein.

The present invention provides a comprehensive 2-D gel database comprising understanding the composition of membrane proteins, in particular 1397 proteins solubilized by NP-40 / UREA and 1191 proteins isolated by SDS / CHAPS / UREA treatment. Builds up the understanding of membrane protein composition in human sperm. To create such a database, a technique was developed to obtain high resolution and to detect a range of acidic, neutral and basic sperm proteins using both IEF and NEPHGE for one-dimensional electrophoretic separation. Recent reports describe the resolution 26 of 680 human sperm proteins in one 2-D gel, while the present invention utilizes a condition in which more than 1000 protein spots are separated in one gel. Improved resolution can be obtained by improvements in sperm purification and solubilization, adjustment of the ampoline composition, and optimization of run time and voltage in both IEF and NEPHGE (see method). In particular, increased voltage during NEPHGE severely affects the resolution of SDS / CHAPS solubilized protein, which results in shorter focusing times and less horizontal streaks than with lower voltages.

Comparison of the sperm protein repertoire solubilized by the two lysis buffers used in the present invention reveals important differences. For example, anionic / amphoteric / urea lysis buffer was needed to solubilize several cytoskeletal components. This extraction method also separated the five 53 kDa isoforms of sperm surface hyaluronidase PH-20, while only three 53 k-Da PH20 isoforms were separated using nonionic / urea lysis buffer. Nevertheless, a nonionic / urea solubilization method was chosen for this study because it can readily solubilize the cell membrane with minimal contamination from the cytoskeletal structure, and the most reliable pI detection methods (eg, FIGS. 5 and 9). In comparison with β-tubulin migration).

44 silver stained gels representing different sperm samples from 8 donors were analyzed by "2-D analyzer" software. Small differences between individuals were observed in the abundance and / or electrophoretic mobility of some proteins. Gene expression, genetic dysplasia, and diversity in post-translational modifications can cause these differences. An example of the latter is phosphorylation at the tyrosine residues of the heterozygous phase of surface proteins having a molecular weight of 89 to 95 kDa. Tyrosine-phosphorylated proteins of similar size have recently been found to have protein kinase activity (35) that is promoted by human ZP-3, suggesting that the protein intervenes in the initiation of zona pellucida binding and peak body reactions. Although differences in phosphorylation and glycosylation can lead to differences between some individuals in the observed electrophoretic shifts, such as capacitation, carbohydrate side chain analysis, protein microsequencing, and analysis of surface changes during cDNA cloning. Is needed before a clear conclusion is drawn on the nature and functional significance of the differences.

It is known that "sperm coated" proteins derived from the discharge of the epididymis, prostate or seminal sac are adhered to the surface of the ejaculated sperm (36, 37). The migration of several seminal plasma proteins was similar to that of sperm proteins in 2-D gels, and nine sperm surface proteins were found to be derived from sperm plasma. In addition, to confirm the origin of surface proteins, analysis of unliquefied seminal plasma proteins (33, 38), different washing and harvesting methods, analysis of epididymal fluid and sperm, and microsequencing of electrophoretically separated proteins were performed. Further assays, including, were devised by the present invention.

Spots of some proteins in the 2-D pattern were identified by immunoblotting or co-migration assays (eg albumin and carboxylic acid anhydrase). Immunological reagent preparations for sperm proteins have revealed a number of proteins in the sperm surface protein dictionary by immunoblotting. Uncertain identification of some sperm proteins was made by comparing the sperm database with other 2-D gel protein databases (16, 39). Moreover, selected surface proteins were purified by 2-D gel electrophoresis and then microsequenced with Edman degradation and tandem mass spectrometry. Conventional unknown protein sequences have been successfully obtained by this approach (Naaby-Hansen et al. In the formulation). Thus, updating the sperm protein dictionary can monitor the understanding of the molecular identity of the sperm protein and help to characterize the protein. Databases of membrane proteins are also useful for studying clinical, genetic and toxic disorders that affect male gametes and / or gamete formation.

181 radioiodinated sperm protein spots were isolated by NP-40 / UREA solubilization and 228 proteins were labeled by surface biotinylation. 98 proteins were labeled by all of the above methods. One interpretation of the different results between radioiodination and biotinylation is that the amino acid residues targeted by the labeling reagents are different. In radioiodination, labeling occurs by electrophilically adding cationic 125-iodine to tyrosine residues and, to some extent, other phenols, histidines, and tryptophan (40), while NHS-LC-biotin is a primary amine group ( Eg lysine residues or free amino groups on carbohydrates or lipids). In addition, steric hindrance by carbohydrate side chains may be interfered by detection by biotinylation or subsequent avidin binding. Assuming that only 60% of the sperm protein is detected by conventionally known silver staining in the somatic cell line 42, approximately 2300 sperm proteins are estimated. This estimated number approximates the number of [ ³⁵ S] methionine labeled proteins (> 3000) found in somatic cells (16, 39) with similar separation methods. Thus, the 311 surface proteins labeled by one or another method are approximately 12% of the total sperm protein. The 98 proteins labeled by iodide and biotinylation include a subset of proteins that are considered first class candidates for microsequencing.

In the method of labeling a cell surface using radioactive iodine or biotin, the principles are: 1) to obtain surface specificity (e.g., to label or not label a small amount of the cell protoplast component) and 2) to accessible residues on the surface ( 43, 44) to obtain a sufficiently specific label. To the sulfonated N-hydroxysuccinimide ester form of biotin (IODO-BEAD) (45) using N-chlorobenzenesulfonamide as oxidant on polystyrene beads (41, 46, 47). Surface specific labeling has been reported previously. In the method of the present invention, all of the known intracellular proteins actin, tubulin, fibrous herb, and intracellular tip protein SP-10 are not labeled by the above two methods, but the labeling method for confirming the surface specificity of the assay It is then important to remove damaged tip responsive cells by Percol density gradient centrifugation.

<Example 13>

Sperm proteins with an apparent mass of 63 kDa and an isoelectric point of 4.3 were separated from other sperm proteins using lysis buffer [NP-40 / urea] and preparative two-dimensional gel electrophoresis (23 × 23 cm gel). Proteins were transferred to PVDF membranes and the membranes stained with Coomassie blue, and the desired protein spots were cut out for Edman microsequencing. For mass spectroscopy, 60 kDa protein spots were taken directly from the prefabricated acrylamide gel using a fine scalp and the protein digested as follows. Two methods of microsequencing were Edman decomposition and tandem mass spectrometry.

Edman decomposition

Protein spots on PVDF membranes were sequenced using cartridges and cycles for PVDF membranes in an Applied Biosystems 470A protein sequencer operated according to the manufacturer's instructions.

Mass spectrometry analysis

The spots were cut as close as possible from the gel to minimize edge polyacrylamide and divided into small pieces. The pieces were destained overnight in 500 μl 50% methanol. Gel pieces were dehydrated in acetonitrile, rehydrated in 50 μl 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at 55 ° C. for 1 hour. The DTT solution was removed and the samples were alkylated in 50 μl of 50 mM iacetamide / 0.1M ammonium bicarbonate in the dark at room temperature for 1 hour. The reagents were removed, the gel pieces were washed with 100 μl of 0.1 M ammonium bicarbonate and dehydrated in 100 μl of acetonitrile for 5 minutes. Acetonitrile was removed and the gel pieces were rehydrated in 100 μl of 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 μl of acetonitrile, the acetonitrile was removed, and the pieces were completely dried by vacuum centrifugation. Gel pieces were rehydrated in 12.5 ng / μl trypsin [blocking autophagy] in 50 mM ammonium bicarbonate and incubated for 45 minutes on ice. Excess trypsin solution was removed and 20 μl of 50 mM ammonium bicarbonate was added. The glands were digested overnight at 37 ° C. and the peptides formed were extracted from polyacrylamide in 200 μl of 50% acetonitrile / 5% formic acid. These extracts were pooled and evaporated to less than 20 μl for LC-MS analysis.

The LC-MS system consists of a Pinningan-MAT TSQ7000 system with a 10 cm × 75 μm id POROS 10 RC reversed phase capillary column and an adjacent electrospray ion source. A 1 μl volume of extract was injected and the peptide was eluted from the column by an acetonitrile / 0.1 M acetic acid gradient at a flow rate of 0.6 μl / min. The electrospray ion source operates at 4.5 kV and uses a coaxial shell flow rate of 1.2 μl / min with 70% methanol / 30% water / 0.125% acetic acid and the coaxial nitrogen flow rate required for optimal sensitivity. To determine the molecular weight of the peptide present in the digest, the digest was analyzed by capillary LC-electrospray mass spectrometry. Peptide sequences for peptides to be detected were determined by collisionally activated degradation using LC-electrospray-tandem mass spectroscopy with argon as the collision gas.

result

Edman decomposition

: 15 N-terminal amino acids were measured by Edman digestion. The obtained sequence EPAVYFKEQFLDGDG (SEQ ID NO: 3) was found to be 100% identical to human calreticulin (GemPep. Accession No. M84739).

Mass spectroscopy

: The molecular weight measured by LC-MS analysis and amino acid sequence measured by LC-Tandum MS of the peptide in the digest is shown in Table 1. Nine peptides were observed and sequence information was obtained for six of the peptides. Database search using CAD spectral information (MSTag) and partial peptide sequence (BLAST) revealed five of the peptides of the human calfpticulin sequence.

Design of Degenerate Oligonucleotides

: Two peptide sequences were selected as templates for designing degenerate oligonucleotides: peptide sequence # 1-EPAVYFKEQFLD (SEQ ID NO: 15) and peptide sequence # 2-KVHVIFNYK (SEQ ID NO: 11). See Figure 12.

FIG. 12 shows amino acid microsequence analysis obtained by Edman digestion and tandem mass spectrometry from sperm proteins of 63 KDa, pi 4.3, selected for the design of complementary and reverse complementary oligonucleotides. From this microsequence analysis, PCR was initiated by synthesizing information pools of degenerate oligonucleotide primers (see FIG. 13).

Protein SEQ ID NO: 1 and its sense-oligonucleotides (SEQ ID NOs: 16-18)

Protein sequence 2 and its sense-oligonucleotides (SEQ ID NOs: 12, 19, 20)

Due to the degeneracy of the genetic code, optimal codons (Lathe, 1985) were used to design "optimized oligonucleotides". Additional parameters involved in the design include: A) regions of protein sequence with minimal degeneracy, reducing complexity of oligonucleotide design, B) preparing degenerate oligonucleotides that include all reasonable sequence substitutions, C) Oligonucleotides were prepared 20-30 nucleotides in length depending on the GC content of the proposed oligonucleotide, the number of reliable amino acid residues sequenced and the intrinsic melting temperature for the particular oligonucleotide pool. Protein sequence # 1 (EPAVYFKEQFLDGD) (SEQ ID NO: 21) shows possible oligonucleotide sequence 5'-GA (A / G) -CC (T / C / A / G) -GC (T / C / A / G) -GT ( T / C / A / G) -TA (T / C) -TT (T / C) -AA (A / G) -GA (A / G) -CA (A / G) -TT (T / C) CT (T / C / A / G) (SEQ ID NO: 17) or TT (A / G) -GA (T / C) -GG (T / C / A / G) -GA (T / C) -3 ' (SEQ ID NO: 22). Using the optimized codons and other parameters described above, the final oligonucleotide sequence was derived as follows: 5'-GC (T / C) -GC (C / G) -TAC-TTC-AAG-GAG-CAG- TTC-CT-3 '(SEQ ID NO: 23). As such, protein sequence # 2 (KVHV (1 / L) FNYK) (SEQ ID NO: 7) is oligonucleotide sequence 5'-CTT-GTA-GTT-GAA-GAT-CAC- (A / G) when optimized and reverse complementary TG-CAC-CT-3 '(SEQ ID NO: 24) was generated. Preparation of oligonucleotide probes was performed by University of Virginia Core Group Facilities.

RT-PCR Cloning Use of Degenerate Oligonucleotides

: 0.05 μg poly- (by _first combining 0.5 μg oligo-d (T) _12-18 RNA and diethylpyrocarbonate (DEPC) treated H ₂ O in 20 μl of reaction before heating at 65 ° C. for 10 minutes. A) PCR cloning was performed by reverse transcription of ⁺ RNA. 2 μl of 10 × RT buffer, 0.5 μl of placental ribonuclease inhibitor (36 U / μl; promega), 1 μl of 10 mM 4dNTP and 1 μl of AMV reverse transcriptase (23 U / μl; stratagen) were added and vortexed, The mixture was incubated at 42 ° C. for 60 minutes. After addition of 80 μl of DEPC-treated H ₂ O, 1 μl aliquots of the cDNA solution were dispensed for 40 times (45 seconds at 94 ° C., 38/44, as specified by Taq polymerase manufacturer (Promega or Perkin Elmer)). Amplification for 45 seconds at / 50/56 ° C for 2 minutes at 72 ° C). Isolation and isolation of PCR products were obtained by electrophoresis of reaction aliquots and fractions of specific fragments in 2.4% agarose gel at 1 × in TAE (40 mM Tris-Acetate, 1 mM EDTA) buffer. After precipitation and quantification, PCR fragments were ligated and sequenced into the pCR-script vector according to the manufacturer's instructions (stratagen).

Degenerate and optimized oligonucleotides were used for PCR reactions using testis cDNA prepared by reverse transcription of poly- (A) -RNA of human testes. PCR reactions were analyzed on agarose gel (FIG. 13) at 38, 44 and 50 ° C. for negative controls (lanes 3, 4, 6, 7, 9, 10, 12, 13 of FIG. 13) performed at the same temperature. A single potent PCR product (lanes 5 and 8 11 in FIG. 13) was demonstrated running at about 400 bps from the reaction at the annealing temperature. Computer analysis showing the electrolysis of the 400 bP band, cloning into the pCR-stript vector, sequencing and sequencing of the bands was identical to the somatic cell calciculin cDNA (Genbank Accession No. m84739) and thus derived from oligonucleotide primers. 2-D gel spots were in the form of the testicular caleticulin (FIG. 14).

FIG. 14 shows DNA sequences (SEQ ID NO: 25) obtained from clones optimized by RT-PCR using optimized and degenerate primers. The numbers indicate the position of base pairs in the sequenced clones and caleticulin genes, (c) represents RT-PCR clones (SEQ ID NO: 25), while (H) represents human calleticulin cDNA from Genbank. (SEQ ID NO: 26).

This example shows successful isolation of human testicular cDNA using amino acid sequence data obtained by microsequencing 2-D gel protein spots.

<Example 14>

Sperm protein I-23 from sperm surface pre {index} [Table III], with an apparent mass of 54 KDa and an isoelectric point of 5.3, was prepared using 23 × 23 cm gel and lysis buffer A [NP-40 / urea] Two-dimensional gel electrophoresis was used to separate from other sperm proteins. The protein was chosen as an example of a double vector labeled cell surface molecule. For mass spectroscopy, I-23 protein spots were taken directly from preparative acylamide gels using fine scalps, the proteins were digested in acrylamide gels and tandem mass spectroscopy was performed as described in Example 13 above.

15 shows protein microsequencing derived by tandem mass spectrometry from sperm surface protein I-23.

15 shows microsequence analysis obtained from protein I-23 in sperm surface index. A) The sequence is a compilation of CAD analysis and CAD analysis of N-terminal derivatives. X represents I or L which cannot be distinguished by low energy CAD. (-) Indicates an unknown amino acid that requires further analysis. (Y / Z) represents another two possible amino acids for the position to be required for further analysis. B) Complementary and Reverse Complementary Oligonucleotides

A) peptide sequence (SEQ ID NOs: 27-35)

Peptide # 1-SPXXSXK

Peptide # 2-XQANNA-K

Peptide # 3-VATEFAFR

Peptide # 4-XSXNXR

Peptide # 5-SMXXDTK

Peptide # 6-MET (x / n) (e / q) XDR

Peptide # 7-VTNSTGGSpxk

Peptide # 8-Uninterpretable

Peptide # 9- --PEVD--tR

Peptide # 10-Uninterpretable

Peptide # 11- --QAENA --- K

B) Protein Sequence 3: Sense Oligonucleotides (SEQ ID NOs: 29, 36, 37)

Protein sequence 7: sense-oligonucleotide (SEQ ID NOs: 38-40)

RT-PCR was performed as described in Example 13, and the product was analyzed on agarose gel as described in Example 13. The main PCR product was approximately 1000 bP. It was cloned into the pCR-script vector and sequenced. The sequence of this novel sperm surface protein is shown in FIG. 16.

Figure 16 shows 1011 bp DNA (SEQ ID NO: 41) obtained from clones optimized for sperm surface protein I-23 and derived by RT-PCR using degenerate primers.

This example shows a method for identifying and isolating novel cell surface vaccinogen cDNA from double labeled cell surface proteins on a 2-D gel, microsequencing, optimizing oligonucleotide design and RT-PCR. . Such combinations have created a rational way of designing vaccines for surface antigens on any biological target, and have paved the way for the design of contraceptive vaccines for related surface antigens, which can lead to antibody immobilization or deadlocking.

The following is a list of documents related to the above descriptions and in particular to experimental and discussion content. The documents should be regarded as cited in their entirety.

references

While the present invention has been described so far, it will be apparent to those skilled in the art that many modifications may occur without departing from the spirit or scope of the invention set forth herein.

(1) GENERAL INFORMATION:

(i) APPLICANT: HERR, JOHN C.

NAABY-HANSEN, SOREN

FLICKENGER, CHARLES J.

WOLKOWICZ, MICHAEL J.

(ii) TITLE OF THE: METHOD FOR THE PRODUCTION OF VACCINES

AGAINST CELL SURFACE PROTEINS

(iii) NUMBER OF SEQUENCES: 41

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT,

P.C.

(B) STREET: 1755 S. JEFFERSON DAVIS HIGHWAY, SUITE 400

(C) CITY: ARLINGTON

(D) STATE: VA

(E) COUNTRY: USA

(F) ZIP: 22202

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patent In Release # 1.0, Version # 1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08 / 806,174

(B) FILING DATE: 25-FEB-1997

(C) CLASSIFICATION:

(viii) ATTORNEY / AGENT INFORMATION:

(A) NAME: KELBER, STEVEN B.

(B) REGISTRATION NUMBER: 30,073

(C) REFERENCE / DOCKET NUMBER: 7762-002-27

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 703-413-3000

(B) TELEFAX: 703-413-2220

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Val Asp Asp Ala Leu Arg Asn Ser Thr Lys Ile Tyr Ser Tyr Phe Pro

1 5 10 15

Ser val

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

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

Gly Pro Ser Leu

One

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Pro Ala Gly Ala Val Tyr Phe Lys

1 5

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Val His Val Xaa Phe Asn Tyr Lys

1 5

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:>

Thr Asp Asn Asn Gly>

1 5>

(2) INFORMATION FOR SEQ ID NO: 7:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 9 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:>

Lys Val His Val Xaa Phe Asn Tyr Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 8:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 11 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:>

Gly Gln Thr Xaa Val Val Gln Phe Thr Val Lys>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 9:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 12 amino acids>

(B) TYPE: amino acid>

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(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:>

Glu Gln Phe Xaa Asp Gly Asp Gly Trp Thr Ser Arg>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 10:>

(i) SEQUENCE CHARACTERISTICS:>

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(B) TYPE: amino acid>

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(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:>

Val Ala Glu Pro Ala Val Tyr Phe Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 11:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 8 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:>

Val His Val Ile Phe Asn Tyr Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 12:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 9 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:>

Lys Val His Val Ile Phe Asn Tyr Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 13:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 11 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:>

Gly Gln Thr Leu Val Val Gln Phe Thr Val Lys>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 14:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 12 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:>

Glu Gln Phe Leu Asp Gly Asp Gly Trp Thr Ser Arg>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 15:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 12 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:>

Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 16:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 11 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:>

Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 17:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 33 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:>

GADCCNGCNG TNTAYTTYAA RGARCARTTY CTN 33>

(2) INFORMATION FOR SEQ ID NO: 18:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 26 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:>

GCYGTSTACT TCAAGGAGCA GTTCCT 26>

(2) INFORMATION FOR SEQ ID NO: 19:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 27 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:>

AARGTNCAYG TNATYTTYAA YTAYAAR 27>

(2) INFORMATION FOR SEQ ID NO: 20:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 26 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:>

AGGTGCAYGT GATCTTCAAC TACAAG 26>

(2) INFORMATION FOR SEQ ID NO: 21:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 14 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:>

Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 22:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 12 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:>

TTRGAYGGNG AY 12>

(2) INFORMATION FOR SEQ ID NO: 23:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 26 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:>

GCYGCSTACT TCAAGGAGCA GTTCCT 26>

(2) INFORMATION FOR SEQ ID NO: 24:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 26 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:>

CTTGTAGTTG AAGATCACRT GCACCT 26>

(2) INFORMATION FOR SEQ ID NO: 25:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 352 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: cDNA>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:>

TTCCCGCTGG ATCGAATCCA AACACAAGTC AGATTTTGGC AAATTCGTTC TCAGTTCCGG 60>

CAAGTTCTAC GGTGACAGAG NAAAANATAA AGGTTTGCAG ACAAGCCAGG ATGCACGCTT 120>

TTATGCTCTG TCGGCCAGTT TCGAGCCTTT CAGCAACAAA GGCCAAACGC TGGTGGTGCA 180>

GTTCACGGTG AAACATGAGC AGAACATCGA CTGTGGGGGC GGCTATGTGA AGCTGTTTCC 240>

TAATAGTTTG GACCAGACAG ACATGCACGG AGACTCAGAA TACAACATCA TGTTTGGTCC 300>

CGACATCTGT GGCCCTGGCA CCAAAAAGGT GCATGTGATC TTCAACTACA AG 352>

(2) INFORMATION FOR SEQ ID NO: 26:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 352 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: cDNA>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:>

TTCCCGCTGG ATCGAATCCA AACACAAGTC AGATTTTGGC AAATTCGTTC TCAGTTCCGG 60>

CAAGTTCTAC GGTGACGAGG AGAAAGATAA AGGTTTGCAG ACAAGCCAGG ATGCACGCTT 120>

TTATGCTCTG TCGGCCAGTT TCGAGCCTTT CAGCAACAAA GGCCAGACGC TGGTGGTGCA 180>

GTTCACGGTG AAACATGAGC AGAACATCGA CTGTGGGGGC GGCTATGTGA AGCTGTTTCC 240>

TAATAGTTTG GACCAGACAG ACATGCACGG AGACTCAGAA TACAACATCA TGTTTGGTCC 300>

CGACATCTGT GGCCCTGGCA CCAAGAAGGT TCATGTCATC TTCAACTACA AG 352>

(2) INFORMATION FOR SEQ ID NO: 27:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 7 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:>

Ser Pro Xaa Xaa Ser Xaa Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 28:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 9 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:>

Xaa Gln Ala Asn Asn Ala Xaa Xaa Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 29:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 8 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:>

Val Ala Thr Glu Phe Ala Phe Arg>

1 5>

(2) INFORMATION FOR SEQ ID NO: 30:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 8 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:>

Xaa Xaa Xaa Ser Xaa Asn Xaa Arg>

1 5>

(2) INFORMATION FOR SEQ ID NO: 31:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 9 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:>

Xaa Xaa Ser Met Xaa Xaa Asp Thr Lys>

1 5>

(2) INFORMATION FOR SEQ ID NO: 32:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 8 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:>

Met Glu Thr Xaa Xaa Xaa Asp Arg>

1 5>

(2) INFORMATION FOR SEQ ID NO: 33:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 11 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:>

Val Thr Asn Ser Thr Gly Gly Ser Pro Xaa Lys>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 34:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 10 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:>

Xaa Xaa Pro Glu Val Asp Xaa Xaa Thr Arg>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 35:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 11 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:>

Xaa Xaa Gln Ala Glu Asn Ala Xaa Xaa Xaa Lys>

1 5 10>

(2) INFORMATION FOR SEQ ID NO: 36:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 27 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:>

GTNGCNACNG ARTTYGCNTT YCGNAGR 27>

(2) INFORMATION FOR SEQ ID NO: 37:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 25 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:>

WCBVMYRMRS BSYYMCSSKM GTNWY 25>

(2) INFORMATION FOR SEQ ID NO: 38:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 7 amino acids>

(B) TYPE: amino acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: peptide>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:>

Val Thr Asn Ser Thr Gly Gly>

1 5>

(2) INFORMATION FOR SEQ ID NO: 39:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 21 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:>

GTNACNAAYA GYACNGGNGG N 21>

(2) INFORMATION FOR SEQ ID NO: 40:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 21 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: other nucleic acid>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:>

GTSACCAACW SCACCGGNGG N 21>

(2) INFORMATION FOR SEQ ID NO: 41:>

(i) SEQUENCE CHARACTERISTICS:>

(A) LENGTH: 1011 base pairs>

(B) TYPE: nucleic acid>

(C) STRANDEDNESS: single>

(D) TOPOLOGY: linear>

(ii) MOLECULE TYPE: cDNA>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:>

TCNNCGNNGG GGGGNNANGC GCGGNANANN GGCTANNNAG GCGGNGANNC NGAGCAGNNC 60>

ATTCGCGGGN GAGGCNNTNN TANAAAANAC NNNTGNANCC NNAATANGCC AAACGCCANA 120>

GGAGGGGNNG NAAANCGGCN NAANNCNCCN NAAAAAANAN NTNCNAANCN CGGNTGGGGA 180>

GAAAATNCCC ATTACAGGNC NTCTNCCCAN NCAGTTTTTT CCAATTACAA GGNGGGGGGN 240>

NGGGNTTTTC NAAAAANGCG CGTTCCCNTT CNGGGGNTNA AAAAAAANAG GGNNGCCTTT 300>

TCCAAGACTN NGCGGGCCCC TTTGGNCTGC ACNANCACGC GGGTCAAAGG CTNNACCGNA 360>

AGGGGGAACC CTAANAGTTT TTCAAGGCCC CAGGGGGNNC GGCCCNGGCT TCAAAAAAGN 420>

CTTGCCAGNA ATGNAGATTC NAACATNNAG AGGAAGNCCA NCCAGGNCCN TCGGGTAAGA 480>

TTAAAAGGGA GGTTTTTTTT CCAAGTTCCN ACGGCCGGCA AGAAAAGGCT NAGGGGCNCN 540>

TGAGNNCCCC CTGGGAGGGG NCCGAAGCCG GCNNGAGGTC ATCCCGGAAA TGGGGGTNGN 600>

AGAGCNGGNA ACAGCAGTGG GNTGAGGCAG GNAGGCAGGG GCCAGCANCA ACAGCAGGAC 660>

AGACTTGANG GCCTCGGGCG NGACAGGGAA GAGGCCCAGC ATGGAGGCGA AGCTGAGGAA 720>

GGCCACGGNA CAGTAGAGGA GCCCGTCTGA GAAGATGANN NAGGCCACGT GCCTNACCAT 780>

GGNGCAGTCC GANAACGGCT TCAAAGTNGC CCCGCGGNAN GTNACAGTAN AATTTGANAT 840>

AGGCACCGGC CACGACCAGG AAACAGAAGG AGNTNATNAT NACCAGGGCC ACGGTGAAGC 900>

CCAGGGATGN NGGCTGACCC TNAGGTGGGG CGTAGGGCAN GNAGAATNGG GNGNCNCAGT 960>

ATTCTCCNAT CTGAGTCCAG GGTATGTGTC NGCCTCCATA TCCTCCCNGT T 1011>

Claims (14)

(a) directionally labeling the protein on the membrane surface, (b) isolating the labeled membrane surface protein by two-dimensional gel electrophoresis, and (c) analyzing the sequence of the isolated membrane surface protein. The method of claim 1, wherein the membrane is on a virus, bacteria or cell. The method of claim 2, wherein said membrane is on sperm cells. The method of claim 1, wherein the membrane protein is labeled with iodine or biotin or mixtures thereof. (a) directionally labeling the protein on the membrane surface, (b) isolating the labeled membrane surface protein by two-dimensional gel electrophoresis, (c) sequencing the isolated membrane surface protein, (d) cloning the DNA encoding the membrane surface protein, and (e) recombinantly preparing a membrane surface protein for use as a peptide immunogen, using the DNA isolated in step (d). The method of claim 5, wherein said membrane is on a virus, microbacterium or cell. The method of claim 5, wherein said membrane is on sperm cells. 6. The method of claim 5, wherein said membrane protein is labeled with iodine or biotin or mixtures thereof. (a) labeling the membrane surface protein obtained in claim 5, (b) reacting the labeled membrane surface protein with serum obtained from a patient to be diagnosed for infertility, and (c) detecting the formation of a complex between the antibody present in the serum and the labeled membrane surface protein. A diagnostic kit comprising the cell surface protein prepared in claim 5. A method of inducing contraception in a patient in need thereof, comprising administering the cell surface protein prepared in claim 5 in an amount sufficient to prevent fertilization of the egg in said patient. A method for producing a contraceptive comprising administering to a mammal the recombinant protein prepared in claim 5 and isolating an antibody against said recombinant protein produced by said mammal. A contraceptive prepared by the method of claim 12. A vaccine prepared by the method of claim 2.