JPH10201493A - Preparation and use of zona pellucida antigens and antibodies for infertility and contraception - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare an antigen useful for inducing the production of an antibody that binds to an epitope on the zona pellucida in an individual. SOLUTION: An immunizing composition and an immunizing method for immunizing an individual to enable production of an antibody against a zona pellucida antigen are disclosed. The use of recombinant molecules and monoclonal antibodies thereto for immunocontraception or infertility has also been disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an individual (individual)
s) Zona pellucida (ZP) antigens and objects for contraception or infertility
The present invention relates to the production method and use of (clonal) antibody. The invention also relates to immunological contraception. More specifically, the invention relates to zona pellucida (zona) produced by recombinant DNA technology.
pellucida) to give an individual autonomic or active immunity to prevent pregnancy using an antigen, or to produce a monoclonal antibody to perform an action against or against the zona pellucida antigen and to passively give the individual ( ) Immunization-related immunological contraception. Further, the present invention relates to a technique for autoimmunizing an individual so as not to become pregnant using an anti-genetic or anti-idiotype monoclonal antibody that mimics the ZP antigen. The present invention further relates to a monoclonal anti-zona pellucida antibody (anti-zona pellucida antibod) which acts against or against a zona pellucida antibody.
y), a novel hybridoma cell that expresses such an antibody, and a method for producing such hybrid cells (hybrid cells) and anti-Zona pellucida (anti-Zona pellucida).

[0002]

BACKGROUND OF THE INVENTION The zona pellucida is a complex extracellular glycoprotein substrate that surrounds mammalian oocytes. This substrate is formed during the early stages of oocyte growth and early differentiation of the follicle (follicle), and serves to protect the oocyte and fetus until implantation into the uterine wall (Cambridge Univ. Press, Cambridge, UK (1982), Austin et al., "Mammalian Reproduction: Germ Cells and Fertilization"). In addition, zona plays an important role in the fertilization process, as fertilization involves the sperm first sticking to the zona and entering the zona. After binding to the zona pellucida of the oocyte, the sperm must penetrate the zona pellucida. Sperm can penetrate the zona pellucida probably due to the limited extent of hydrolysis of zona pellucida elements by sperm enzymes such as acrosin (Ann. Rev. Biochem. 43: 7).
77 (1974); McCrawley et al .; Biol. Reprod. 32: 619 (198
5), Dunbar et al.); Gam. Res. 1:65, 1978, Stanbau). The zona pellucida remains intact after fertilization, which allows the fetus to grow well and prevent fetal fusion in the fallopian tubes (Science 138: 594, 1962, Mintz). Finally, the zona pellucida acts to prevent large numbers of sperm from being fertilized.
When fertilized, some mammals alter sperm binding to the zona pellucida, resisting proteolytic digestion
(J. Exp. Biol. 33: 358, 1956, Austin and Braidon).

[0003] Compared to primates close to humans and other species including humans, the properties of rodent zona pellucida are primarily biological, morphological, physiological and immunochemical. Changes to See Academic Press, p.
p.139 (1983) Dunbar, New York, "Mechanisms and Control of Fertilization (J. Hartmann, ed.)"; Oxford Univ.
Press, pp. 505 (1983), Dunbar, London, International Conference on Reproductive Immunity (Wegmann and Gill III, ed.
s.) "); Modern Cell Biology 3 (Satir, ed.) Alan R.
Liss, pp. 77 (1984), New York, Dunbar et al. In mammalian zona pellucida, the major glycoproteins are composed of a limited number (Dev. Bi
ol. 76: 185 (1980), Braille et al .; Biol. Reprod. 24:11.
11 (1981), Dunbar et al .; Biol. Reprod. (1987) Timmons and Dunbar). Different species have different structural and functional relationships in the zona pellucida.

[0004] Suppression of fertilization of oocytes by sperm,
Insufficient materials (substances) to produce a significant amount of antigens and antibodies needed to create a vaccine that prevents implantation of fertilized eggs or prevents ovarian development, thus making immunocontraception effective and economical Could not be done. Early immunocontraception methods have been largely unsuccessful. This method used naturally occurring circulating peptides such as chorionic gonadotropin (hCG) and follicle maturation hormone (Ple
num Press (GP Talwan), 1986 New York, Griffin "Implications for Contraception and Promotion of Fertility". Usually, the use of antibodies that resist "circulating" antigens in immunocontraception creates the undesirable problem of immune complexes damaging tissues. Furthermore, it was also found that immunization using the "circulating" antigen had little effect on suppressing fertilization.

[0005] Immunologically based methods of contraception are more desirable than commonly used surgical sterilization methods or fertility control pills (for humans and pets). In the case of pills, they have to be purchased and taken at regular intervals, so the cost of medicine is constantly high and the effect is only temporary. Therefore, there is a need for an antigen that is safe, highly reliable, economically provided at low cost, and can temporarily or permanently contracept an individual.

[0006]

The present inventors have focused on the use of purified or purified zona pellucida for immunological sterility or contraception. Providing immunity with zona pellucida antigens has distinct advantages over other immunological contraceptive methods. Immunization with the zona pellucida suppresses fertilization, not the effect of ovulation. This method of immunocontraception is particularly preferred for humans. In addition, this method can also reduce the development of ovarian follicles and render them permanently infertile. Such permanent sterilization of pets and other animals can be performed non-surgically, without the risks and costs associated with conventional surgical procedures.

Another advantage of zona pellucida protein contraception is that fertilization can be prevented with a few liters of zona. This is due to the action of the immunocontraceptive antibody exhibiting locally unique properties at the site and limiting the naturally occurring amount of zona pellucida protein in individuals seeking to prevent fertilization. Previous work on the tissue properties of zonal antigens has been on the ovaries, where the antigens do not circulate. In contrast, hormonal proteins circulate throughout an individual and occur at much higher levels. The level of circulating antigens also varies greatly depending on the physiological state of the individual.

[0008] Furthermore, the level of sperm antigen required for effective contraception of sperm antigens varies depending on the amount of sperm in the vaginal canal and uterine cavity. Because the ZP proteins of various animals are immunologically crossreactive, there is no need to develop immunizing antigens in the animal to be contraceptive.

One object of the present invention is to provide a method for effective contraception by an immunological method. In this way, only one (or minimal) dose need be administered, and there is no need to be constantly involved with a physician (in the case of humans) or a veterinarian (in the case of animals). Also permanently infertile or castrated
(Preferable for pets) or temporarily infertile (desirable for humans and bred pets).

[0010] Also, one of the disadvantages of the conventional method is the limited supply of zona pellucida antigens and antibodies for use in immunocontraception. In contrast, the present invention provides a method for obtaining zona pellucida antigens and antibodies for performing immunocontraception, such as zona pellucida antigens, monoclonal zona pellucida antigens and ZP anti-genotype antibodies produced by recombinant DNA technology. Is extremely abundant. Because antigens and antibodies can be produced in large quantities using recombinant DNA technology and hybridoma technology,
There is the added advantage of low cost.

The present invention is directed to the preparation of antigens and a method of immunizing animals to induce antibodies that react with determinants of epitopes found in zona pellucida antigens. The present invention further relates to monoclonal anti-Zona pellucida antibodies, novel hybridoma cells that express such antibodies, and methods of making such hybrid cells and anti-Zona pellucida antibodies. Furthermore, the invention relates to monoclonal antibodies with anti-genetic zona pellucida and to the use of such antibodies for autonomic or active immunocontraception.

An antigen of an individual (for example, glycoprotein (glycop
rotein)) are a number of antigenic determinants, or "epitope"
This factor is recognized by the antibody (rec
ognize). Epitopes include amino acid sequences, carbohydrate residues, conformational or “shape”
Or the site at which two different molecules or peptides interact. The zona pellucida glycoprotein contains all these determinants (Bio. Repr.
o. 30: 445 (1984), Drell and Dunbar: Biol. Repro. 3
6: 1275 (1987), Timos).

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In a first embodiment of the present invention, an antigen containing a polypeptide portion of a zona pellucida protein antigen is produced by using a recombinant DNA technique. In an embodiment of the protein of the present invention, an antigen containing the polypeptide determinant portion of zona pellucida protein is produced using recombinant DNA technology. These recombinant polypeptides and their homologs (analogs) are collectively referred to herein as recombinant zona pellucida proteins.

The term "zona pellucida protein" is intended to include polypeptides having the same amino acid sequence as a naturally occurring zona pellucida protein, a recombinant zona pellucida protein and its homologues. .
The term "homolog" refers to a zona pellucida protein by the addition, deletion or substitution of one or more amino acids, provided that the polypeptides are substantially the same in antigenic and biological activity. Include different proteins or polypeptides. These homologs contain selected determinant sites for zona pellucida proteins.
Using these antigens, animals can be immunized to produce antibodies. In the present invention, the antigenic agent of the present invention (antigen preparation) and a drug composition having an immune response enhancing element,
It is included in the present invention, together with a pharmaceutically suitable carrier. Thus, in one embodiment, the present invention provides:
An almost purified polypeptide containing the amino acid sequence of the zona pellucida protein or its part, an expression carrier (e) containing a DNA sequence encoding the zona pellucida protein.
xpression vehicles), hosts modified with such expression vehicles, methods of making zona pellucida proteins in the host, and immunization of the animals with drug compositions having zona pellucida recombinant proteins. And methods for inducing the production of antibodies to band antigens.

In another embodiment, the invention includes a monoclonal anti- zona pellucida antibody, a novel hybridoma cell that expresses such an antibody, and a method for immunological contraception using such a monoclonal antibody.

[0016] By fusing myeloma cells and spleen cells from immunized donors, a homogeneous
(homogeneous) antibody. Thus, a genetically stable hybridoma cell,
A continuous cell line of hybridoma cells has been developed that can produce large amounts of monoclonal antibodies against malignant tumors and specific viruses and their antigenic determinants.

US Pat. No. 4,172,124 to Koprowski and others discloses a specificity for malignant tumors.
Antibodies showing (specificity) may be produced by somatic cell hybrids between malignant tumors deficient in hypoxanthine phosphoribosyltransferase and spleen or lymph cells obtained from animals previously injected with tumor cells. Have been described. Furthermore, U.S. Pat. No. 4,196,265 to Koprowski and others discloses a genetically stable fused cell hybrid, which provides a large amount of monoclonal antibodies against specific viruses and their antigenic determinants. It has been described that a continuous cell line of a hybrid that can be made can be made.

By using such a cell fusion technique, an anti-Zona pellucida antibody, for example, an immunocontraceptive antibody can be stably supplied, and the reliability is high. Individual immunity is active
(Automatically) or passively. The term "active (autonomous) immunity" means that an antigen or immunogen is administered to an individual and that the individual's immune system makes the individual resistant to the antigen. As the immunizing antigen, any substance can be used as long as an individual can produce an antibody. In the present invention, in order to render an individual contraceptive or infertile, an antigen effective for autoimmunizing the individual includes, regardless of whether it is glycosylated (glycosylated) or not, is produced by recombination. ZP antigen (rZP) and anti-genotype ZP antibodies. The term "passive immunization" means that antibodies produced in vitro outside the individual are administered to the individual for immunocontraception. The ZP monoclonal antibodies of the present invention, when administered to an individual, create such passive immunity.

Passive immunization of a ZP antigen with a monoclonal antibody results in temporary (preferably several months) sterility. The anti-ZP monoclonal antibody suppresses fertilization by interfering with sperm that binds or invades the zona pellucida, and does not adversely affect ovarian function at this time.
As described above, the immunocontraceptive method of the present invention can be carried out passively by administering a monoclonal antibody that reacts with the zona pellucida antigen, or can be carried out using a zona pellucida antigen or an anti-zona pellucida produced by recombinant DNA technology. It can also be performed automatically by administering a genotype monoclonal antibody. When using a homogeneous antibody (eg, a monoclonal antibody) as the antigen, a portion of the molecule is responded to by the immunized host and recognized as an antigenic determinant. The unique combination site of a homogeneous antibody that recognizes the antigenic determinant is referred to as "genotype".
For this reason, antibodies made against these sites of the antibody are termed "anti-genotypes or anti-genotypes." These antibodies may have `` internal images '' or have activity mimicking the original immunogen (Proc.Soc. Natl.Acad.Sci. (USA) 75: 2443 ( 19
78), Sage, Kay and Peterson PA: Proc. Soc. Nat
l. Acad. Sci. (USA) 77: 7385 (1980), Shriver et al.).

As shown in FIG. 8, the monoclonal antibody (PSI) inhibits sperm from binding to the surface of the zona pellucida.
Recognize the carbohydrate component of P. PSI monoclonal antibodies can be used as immunogens to generate anti-genotype antibodies. Antibodies can be used for immunological sterility or contraception. Determinants that bind to monoclonal anti- Zona pellucida antibodies can be cloned and modified using recombinant DNA technology to create "single-chain antibodies" against the ZP antigenic determinant (Proc.
Acad. Sci. USA 81: 3273 (1984), Cabilly et al .; Nucl.
eic Acids Research 12: 3791 (1984), Boss et al.).

At its most basic level, the invention discloses the genetic design and preparation of zona pellucida antigens, as well as fertilization, implantation, follicular development and subsequent ovarian hormone formation. The purpose of the present invention is to elucidate the use of said antigens to stimulate the immune system of animals and to induce the formation of antibodies against zona pellucida antigens with the aim of preventing their function.

The present inventors have devised a method for producing cDNA encoding zona pellucida antigen using recombinant DNA technology. ZP cDNA is expressed in the expression vector λ.
gt11. Escherichia coli ( E. coli ) is transformed using the expression vector. The ZP protein or its determinant that expresses the clone is identified by ZP antibody binding. DNA isolated from single-stranded phage was used to express DNA copies (transcripts) and cDN to express ZP antigen.
Used as a template to create A. ZP
Λgt11 phage DNA encoding DNA sequence
Is inserted into the pEX vector. Using pEX,
Transforming the bacterial host into which the DNA is to be expressed,
It produces a large amount of ZP antigen that can be used for immunocontraception.

To determine the partial DNA sequences of the three 9 cDNA expression clones selected by screening the λgt11 expression library with affinity purified ZP antibodies, the M13 cloning method (Proc. Acad. Sci. USA 74: 5463 (1977), Sanger et al.). That is, the cloning method is a dideoxy chain termination reaction method, and zona pellucida DNA is cloned in a filamentous bacteriophage M13.

When the zona pellucida gene is expressed in an E. coli host with a nucleus, the polypeptide produced is not glycosylated. Thus, the molecular weight of the main porcine ZP polypeptide is about 35,
55 and 80 Kd. Also, the molecular weights of the major rabbit ZP polypeptides are 50, 75, 85 Kd (Table 1), lower than those observed for glycosylated molecules (Table 1). For example, among prokaryotes, zona pellucida protein produced from zona pellucida DNA with the code for zona pellucida polypeptide is referred to as "rZP" or "recombinant zona pellucida". Furthermore, when a gene having the code for zona pellucida protein is produced in eukaryotes, the protein is glycosylated, and the glycosylated protein is called "rg
ZP ". “Immunologically relevant antigens”
Antigens with significant genomic homology to zona pellucida proteins are meant, and the products expressed by these DNAs are immunologically cross-reactive and show significant levels. Examples of such immunologically relevant antigens include polypeptides that contain fewer amino acids than the naturally occurring ZP antigen. It has cross-reactivity with zona pellucida polypeptide, an immunologically or biologically important property.

The term "host" as used in the present invention is intended to include not only prokaryotes but also yeast (yeast) and filamentous fungi, as well as plant and animal cells. “Prokaryotes” shall include all bacteria that can be transformed with DNA or rZP protein to express zona pellucida. "Eukaryote" shall include all yeast, fungi, animal and plant cells that can be transformed with DNA or rZP protein to express the zona pellucida.

Zona pellucida protein DNA can be obtained from any mammal. All that requires that the glycoprotein gene sequence be expressed in a prokaryotic or eukaryotic organism. It is particularly desirable that the zona pellucida DNA sequence be immunologically cross-reactive between mammals (eg, pigs, rabbits, dogs, cats or humans).

When changing the host using any technique known to those skilled in the art, a recombinant DNA molecule having a zona pellucida protein code can be used.
For conversion of prokaryotes, it is particularly desirable to use a vector containing a sequence with a zona pellucida code.

The zona pellucida recombinant protein (rZP) of the present invention
Can have some amino acids at its flanking ends as compared to the amino acid sequence of naturally occurring, ie, natural zona pellucida.

"Substantially pure"
When the term is applied to a zona pellucida protein of the present invention, the polypeptide may have a normal association with the zona pellucida protein in its native state, or may be capable of constantly growing electrophoretic or chromatographic responses, Elution profile (eluti
on profiles), and exhibit no antigenic activity. The term "substantially pure" does not exclude artificial or synthetic mixtures of zona pellucida proteins with other compounds.

[0030] Methods for making linking genes by fusion and expressing these genes into bacteria are known and are disclosed, for example, in US Patent No. 4,366,246. The gene production and methods described herein can be used to perform zona pellucida protein expression in prokaryotic or eukaryotic hosts.

The prokaryotic host may be a Gram-negative or Gram-positive bacterium, for example E. coli, S. Tymphimur
ium , Serratia genus Marsessen and Bacillus subtilis.
The eukaryotic host is Pichia pastoris
s) or yeast such as mammalian cells.

In general, an expression vector contains a promoter sequence for efficient transcription of an inserted DNA fragment, and the vector is used together with a host. Expression vectors are
It usually contains an origin of replication, a promoter, a terminator, and also contains a specific gene capable of performing expression selection in a transformed or transformed cell. The transformed host can be grown into optimal cells by fermentation and culturing by means known in the art.

Examples of promoters that can be used in the present invention include, but are not limited to, rec A, trp, lac, tac, and bacteriophage lambda pR or pL. Examples of plasmids or bacteriophages that can be used in the present invention include those listed in "Molecular Cloning" by Moniatis et al. (Cold Spring Harbor Laboratories, 1982), but those skilled in the art In addition to this, an appropriate one can be used.

The present invention applies to any host modified by the methods described above, and to any host modified by methods generally known to those skilled in the art. To produce prokaryotes or eukaryotes that express the zona pellucida protein gene. As a method known to those skilled in the art, for example, a method of transferring a genetic substance using a lysogenic phage is exemplified.

A gene is a DNA sequence that encodes a characteristic amino acid sequence for a specific peptide through its template or messenger RNA. The term cDNA includes
It shall include genes from which intervening sequences have been removed. rDN
The term A means a molecule that has been spliced in vitro (in vitro) with a cDNA or genomic DNA sequence and recombined.

A cloning vehicle, or cloning vehicle, is a plasmid or phage DNA or other DNA capable of replicating in a host cell, characterized by one or a few endonuclease recognition sites. At that site, such a DNA sequence or the like can be trimmed to provide the determinant without compromising the essential biological function of the DNA. The vehicle also contains suitable markers used to identify transformed cells. Markers include, for example, tetracycline resistance or ampicillin resistance.
The term "vector" is sometimes used as a cloning vehicle. Expression vehicles are similar to cloning vehicles, but usually have the ability to express a structural gene in a host while under the control of regulatory sequences.

Hosts transformed with the zona pellucida genome with zona pellucida proteins are particularly useful for the production of zona pellucida polypeptides and proteins and can be used for immunization of animals. As already explained, glycosylation does not occur when the zona pellucida protein genome is expressed in bacteria. For this reason, the molecular weight of the full-length recombinant zona pellucida protein is smaller than the natural or native molecule.
(See Table 1). The recombinant zona pellucida protein may be the entire amino acid sequence of the zona pellucida protein or only a specific determinant. Animals immunized with the zona pellucida recombinant protein produce antibodies that bind to epitopes present on the recombinant or native polypeptide. In this way, recombinant proteins containing zona pellucida can be made on a commercial scale.

"Immunologically effective amount" used in the present invention
By shall refer to the amount of zona pellucida antigen required to make an antibody that binds to the zona pellucida epitope in the animal. The term "individual" includes any animal, but preferably includes mammals, most preferably cats, dogs, cows or humans.

The zona pellucida recombinant proteins of the present invention are particularly useful for sensitizing animal immune systems. One consequence of this is that antibodies are produced that react with epitopes present in the zona pellucida.

The immunization of an individual can be performed autonomously or passively. The term "autonomous (active) immunity" means that an antigen or immunogen is administered to an individual and that the individual's immune system makes the individual resistant to the antigen. As the immunizing antigen, any substance can be used as long as an individual can produce an antibody. In the present invention, in order to render an individual contraceptive or infertile, an antigen effective for autoimmune immunization of the individual includes ZP antigen produced by recombination irrespective of whether or not it has a glycosidic bond. (rZP) and anti-genotype ZP antibodies. The term "passive immunization" means that antibodies produced in vitro outside the individual are administered to the individual for immunocontraception. When the ZP monoclonal antibodies of the present invention are administered to an individual, such passive immunity is created.

If an individual is immunized with a ZP antigen or an anti-genotype antibody before ovarian maturation, ovarian development is severely inhibited and irreversible permanent sterility can be achieved. By this method, animals can be immunized and contraceptive, and it is particularly desirable to apply them to animals such as cats and dogs that do not want to be reproductive. Such ZP
In the case of immunocontraception, costly and potentially dangerous surgical procedures are not required. For immunocontraception, zona pellucida, which immunologically cross-reacts with other species, is desirable. DNA encoding zona pellucida obtained from pig or rabbit ovaries is preferred.

In another embodiment, the present invention discloses a novel continuous hybridoma cell line that expresses a zona pellucida monoclonal antibody and further discloses the use of such a cell line for the production of antibodies, and the production of such a cell line. To The present invention further discloses a method for obtaining large quantities of anti- zona pellucida antibodies used for passive contraception.

According to the present invention, to obtain a novel continuous hybridoma cell line which expresses anti-Zona pellucida antibodies,
Animals and myeloma cells immunized with a zona pellucida protein, preferably a zona pellucida recombinant protein, most preferably a native zona pellucida antigen, and immunized with hybrid cells fused between antibody-producing cells , A hybrid clone, and a clone that expresses the anti-Zona pellucida antibody. More specifically, purified zona pellucida antigen is injected into a mouse or other animal, and the antibody-producing cells of the animal's spleen are fused with cancer-type mouse cells or myeloma cells. Clones of cells producing such antibodies are selected and grown as continuous cell lines. From this, a large amount of anti-Zona pellucida antibodies can be obtained.

Alternatively, the hybrid cells of the clone are
It can be injected and grown in histocompatable animals to produce high levels of anti-Zona pellucida. Cells can be recovered from ascites fluid in the animal. Thus, the present invention provides anti-zona pellucida antibodies used for immunocontraception on a fairly large scale.
This enables stable and reliable supply. The term "immuno-contraception" comes from an immunologically-intervened method, and refers to temporary contraception that can be fertilized later, permanent contraception that cannot be fertilized again,
Or infertility.

The present invention relates to the use of monoclonal ZP antibodies for passive immunization to effect temporary infertility or to use natural antigens (Immunological Reagents) for active immunization.
v. 94:25, 1986, Arranger et al.) including the use of anti-genotype antibodies that mimic the structure.

The administration to the zona pellucida recombinant protein and the monoclonal antibody can be performed by injection or by using a long implant (l).
ong release implants), rapid infusion,
Parenteral administration, such as intravenous or nasal absorption, skin absorption, and oral administration can be used.
Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, and the like. Closed bandage
(occlusive dressings) carriers can be used to increase skin permeability and enhance antigen absorption. The liquid form for oral administration is generally a liposome solution containing the liquid dosage form. Suitable forms of liquid administration include emulsions, suspensions, solutions, syrups, elixirs and the like. These include inert diluents generally known in the art, such as, for example, clean water. In addition to the inert diluents, auxiliary substances such as adjuvants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents and flavoring agents and the like can be further included.

In the case of an antigenic preparation containing the zona pellucida recombinant protein according to the present invention, an adjuvant can also be included. Adjuvants are substances used to nonspecifically promote a specific immune response. Usually, the adjuvant and the antigen are mixed before being given to the immune system or are given separately. However, it is given to the same site of the animal to be immunized. Adjuvants are loosely divided into several groups, depending on their composition. These groups include:
Oil adjuvant (complete adjuvant and incomplete Freund's adjuvant), mineral salts (e.g., AlK (SO _{4) 2,} AlNa
(SO ₄ ) ₂ , AlNH ₄ (SO ₄ ), silica, alum, Al (OH) ₃ , Ca ₃ (P
O ₄ ) ₂ , kaolin and carbon), polynucleotides (eg, poly IC acid and poly AU acid), and natural substances (eg, wax D from Mycobacterium tuberculosis, as well as Corynebacterium parvum ). Substances found in
Pertussis and part of Brucella spp.).

By using the zona pellucida antigen preparation of the present invention in an animal body, an antibody that binds to a zona pellucida epitope determinant can be produced. A particularly useful method for improving the production of antibodies to the zona pellucida is to firstly immunize an animal with an antigenic preparation of the zona pellucida according to the invention and then to a second immunization. is there.

The first immunization is of age of the animal. For permanent immunocontraception or infertility, it is most desirable to immunize the animal at the age of two to three months before the onset of adolescence. This is because it most significantly interferes with ovarian maturation. To contraceptively restore fertilization, express Z after follicle development in the ovary.
P antigen may be used.

There is a method for determining whether or not an animal has been immunized by judging the immune status of the animal against the zona pellucida antigen. In this evaluation, an antibody against the zona pellucida can be detected by using an immunoassay for the zona pellucida recombinant protein according to the present invention. As an immunoassay, for example, an ELISA assay (Biol. Reprod. 30: 445, 1984,
Dorell and Dunbar) are known. In that case, it is possible to determine when the titer of the individual antibody to the zona pellucida is sufficiently high, ie, when immunity can be reliably achieved and pregnancy can be prevented.

Many immunization regimens
When is used, there are many techniques that depend on the timing of the immunization procedure. It is possible to use the antigenic preparation according to the invention more than once, thereby increasing the level of expression of the repertoire of immunoglobulins expressed by the immunized animal and increasing the diversity be able to. In the case of the multiple immunization method, an interval of 1 to 6 months is generally provided.

The appropriate amount of the zona pellucida recombinant protein administered to an animal varies depending on the age and condition. It also depends on whether the purpose of the immunization is for contraception or for castration of the ovaries, and for other factors as well. However, the skilled artisan can readily ascertain and adjust the amount to be administered.

The administration of the antigenic preparation according to the present invention comprises:
It may be performed multiple times or multiple times. The dose of the zona pellucida antigen can be varied in the range of 0.01 to 5 g / ml at a time. In addition, more desirably, 0.05 to 1.0 g / ml,
Most preferably, it is 0.1 to 0.5 g / ml.

Having described the overview of the present invention, the present invention can be more completely understood by referring to the following specific examples. It should be noted that these examples are illustrative, and should not be construed as limiting the present invention unless otherwise specified.

[0055]

EXAMPLES Example 1 Separation of ZP Protein and Preparation of Polyclonal Antibody Isolation of the Zona (ZP) The zona (ZP) was isolated according to the method of Wood et al. Described in Biol. Repr. 25 (2): 439 (1981). A device equipped with two wheels (diameter: 10 cm) and having razor blades arranged in 50 rows each separated by a stainless steel washer (2 mm) was used. One of the two wheels was fixed, and the other was attached to a rotating handle via a chain. The ovaries were dropped between the razor blade wheels immersed in a resin glass tank. In addition, pH7.
2 liters of 0.01 M phosphate buffer (PBS), 2 mM sodium citrate, and 2 mM EGTA. After passing about 300 ovaries through a razor blade wheel, the ruptured ovaries were removed from the bottom of the resin glass tank by 10
Deposited on a removable nylon screen of 00 μm mesh. The screen was removed to separate the ovaries from the buffer and washed thoroughly to remove the oocytes attached to the ovaries. The oocytes and zona pellucida were then separated by sieving through nylon mesh screens of various sizes. Oocytes were homogenized and washed through a 50 micron mesh nylon screen to retain the stratum. When using this method, about 100,000-300,0 in 8 hours
00 ZP could be separated.

B. Purification of ZP protein (purificatio
n) ZP proteins were purified (purified) using two-dimensional polyacrylamide gel electrophoresis (PAGE) with high resolution. PAGE is described by Danbar et al. In Biol. Repr. 24: 1111 (1981), as described above. The method separates proteins by isoelectric focusing in the first dimension and polyacrylamide gel electrophoresis (PAGE) in the second dimension to separate sodium dodecyl sulfate (SDS).

Approximately 30,000 pellets of the stratum were mixed with 300 μl of solubilization buffer, 2% SDS, 2% β-mercaptoethanol, and 1% cyclohexylaminosulfonic acid.
(CHES) and resuspended in water at pH 9.5-9.6. Forty microliters were added per 4% PAGE (containing 1.8% bisacrylamide cross-linker) gel. Amphorine (amphol) with a wide pH range of 3.5 to 10
ines) (LKB) was added to the PAGE gel. Gel is 200
After preliminarily focusing at V for 2 hours, focusing was performed at 25 ° C. and 400 V for 16 hours.

For the second dimension SDS PAGE, a slab gel (10-20% gradient polyacrylamide and 0.8% bisacrylamide crosslinker) was used. The gel was electrophoresed at room temperature at about 3 amps with the dye tip reaching the bottom of the gel. Proteins can be identified by either coomassie blue or silver by staining methods. ZP antigen
Due to the progress of glycosylation, it has a unique protein profile that is heterogeneous in both charge and molecular weight. After transfer to nitrocellulose by electrophoresis as described below, the ZP antigen was immunoblotted as shown in FIG.
t). ZP protein was eluted from the gel by electrophoresis. The N-terminal sequence of the ZP protein was determined by the method disclosed by Hank Pillar et al. In Meth. Enzymol. 91: 227 (1983).

The molecular weight of the major porcine ZP polypeptide is about 35, 55 and 80 Kd. Also, the main rabbit Z
The molecular weight of the P polypeptide is 50, 75, 85 Kd (Table 1), lower than the value observed for the glycosylated molecule (Table 1).

[0060]

[Table 1]

Example 2 Preparation of Polyclonal ZP Antibody A polyclonal antibody was applied to female rabbits or castrated ewes by 300-500 μg of a zona pellucida (HSZ), all of which were solubilized by heat.
P) (60-65 ° C in 0.01 M sodium carbonate buffer, pH 9.
5 for 1-2 hours). Alternatively, purified ZP separated on a 2D-PAGE gel
Immunization was performed with approximately 50-200 μg of protein. All samples of the protein were suspended in 1 ml of water and emulsified with Freund's complete adjuvant before injection. Many sites were injected into the animal's cortex under the scapula and 4 weeks later boosted in incomplete Freund's adjuvant with half the initial immunization dose of ZP protein. For some, additional boosts could be performed monthly with Freund's complete adjuvant to obtain higher antibody titers.

The antibodies were analyzed and characterized using an enzyme-linked immunosorbent assay (ELISA) or immunoblotting. EL
In the ISA assay, rabbit, sheep or mouse immunoglobulin was detected using a vector dye kit (Vecta Stain Kits) obtained from Vector Laboratory. For this assay, 50-100 μg of 0.1 M NaCO ₃ , pH 9.0 HSZP protein was added into the wells of a 96-well titration tray. Next, an assay was performed by a known method based on the instructions of the kit. In immunoblotting,
Specific ZP glycoproteins and peptides were identified using the method disclosed by Timmons et al. In Biol. Repr. 36: 1275. An unstained, unfixed 2D-PAGE gel was placed on the cathode side of nitrocellulose paper (Bio-Rad) and 2.5 A at 1.3 A using an EC electroblot transfer unit.
Time transfer was performed. The nitrocellulose paper is then treated with 3% by weight bovine serum albumin (BSA) and 0.01% sodium azide in 10 mM tris (hydroxymethyl) aminomethane (tris) -saline, pH 7.2. , Blocked overnight. Then, Tris-brine-
The azide (without BSA) was changed twice and washed. Polyclonal serum (1-5 ml) or monoclonal antibody supernatant (100 μl)
g / ml of immunoglobulin G (IgG) was diluted in 50 ml of Tris-brine-azide (BSA) and incubated with nitrocellulose transfer with shaking overnight at 25 ° C. After that, Tris-brine-azide (without BSA) was changed twice and washed.

Next, the nitrocellulose transfer was cultured with ¹²⁵ I-protein A for polyclonal serum or ¹²⁵ I-goat for monoclonal supernatant and anti-mouse IgG. 10 ⁶ cpm total in 50 ml Tris-brine-azide
Was added to each transfer and cultured overnight with shaking. Before air drying, the plate was thoroughly washed in Tris-brine-azide (BSA), exposed to Kodak XAR-5 X-ray film, and radiographed.

The antibody was a CNB conjugated to the ZP protein.
Affinity cleaning was performed using r-activated Sepharose. CNBr activated Sepharose 4B resin (Pha
rmacia) was used to prepare the affinity column. The resin was bound to either heat solubilized HSPZ or HSRZ (rabbit heat solubilized zona pellucida). About 1mg / ml ZP
The preparation was made in a solubilization buffer at pH 9.6, as described above. ZP is a binding buffer (0.1 M NaHCO ₃ ; 0.5 M Na
Cl, pH 8.3). Wash the resin, 1mM H
Swelled in Cl and washed quickly with binding buffer before placing ZP into binding buffer. Buffers include:
Includes 0.5 NaCl to minimize protein-protein interactions. The mixture rotated slowly at room temperature for about 2 hours. Binding buffer was removed along with unreacted ZP solution, and the resin was incubated in 1 M ethanolamine overnight at 4 ° C. with slow rotation to occlude the remaining active groups. Next, the resin was 1 mM NaHCO ₃
(pH 8.3, 0.5 M NaCl) three times, followed by 0.1 M acetate buffer (pH 4, 0.5 M NaCl).

The antiserum was incubated on the bound resin at 4 ° C. with gentle rotation overnight. Prior to elution, the resin was warmed to room temperature and washed thoroughly with borate / salt buffer (100 mM boric acid, 75 mM sodium borate, 75 mM NaCl, pH 8.4) to completely remove unbound protein. The bound IgB was eluted with 200 mM glycine (pH 2.7, 0.8% NaC).
This was performed using l). The acid fraction was collected directly in 0.2 M Trizma base and the final solution was Trizma: Glycine 1: 2, pH 7.5. This neutralizing action can be eluted to minimize damage to the highly purified antibody. Aliquots of the fractions containing the antibody were aliquoted and frozen for analysis. Cleaning of the immunoaffinity antibody was revealed by one-dimensional SDS-PAGE analysis.

Example 3 Isolation of ZP Protein Expressing cDNA Clones A person skilled in the art can use a method disclosed by Chilgwin et al. In Biochem. 18: 5294 (1979) to prepare a 6-week rabbit and a 12-week rabbit. It can be appreciated that RNA can be prepared from the ovaries of rabbits for more than 6 months. The ovaries contain a large amount of connective tissue, so homogenization (homogen
It is desirable to freeze in liquid nitrogen and then crush to a fine powder before ization). 4-8 ovaries were homogenized in 4M guanidinium thiocyanate (0.025M sodium citrate, 0.5% by weight sodium lauryl sarcosine, pH 7.0) in the presence of mercaptoethanol. The homogenate is one of the JA-20 rotors.
It was centrifuged at 10,000 rpm for 10 minutes at 0,000 rpm. All RNA was separated from the protein by ethanol precipitation or sedimentation through cesium chloride. The supernatant layer is 5.7 M CsCl containing 3.8 g / 100 mls of EDTA (disodium salt) and in a SW-40 rotor,
Centrifugation was performed at 000 rpm at 20 ° C. for 20 hours. The pellet was resuspended in guanidine hydrochloride. RNA is
0.3 ml of 3M sodium acetate (pH 5.2) and 95% ethanol
Precipitate at 2.2 volumes. After overnight culture at -20 ° C, the RNA is recovered by centrifugation at 11500 rpm, -5 ° C for 10 minutes. RNA is 95
Washing was performed twice with% ethanol and dissolved in water.
RNA recovery is monitored by measuring the 260/280 absorbance ratio. Polyadenylation
nylated) RNA was separated using oligo (dT) cellulose chromatography. This chromatography is
Cold Spring Harbor Laboratory (1982), p. 197, in Molecular Cloning by Maniatis et al. The fraction of the chromatography column is I
Monitored at A ₂₆₀ using an SCO spectrophotometer.

Cell type specific cDNA probes are available from Proc.
Natl. Acad. Sci. USA 81: 2194 (1984), Davis et al., And Proc. Natl. Acad. Sci. USA 80: 1194 (1983), Young et al. FIG. 3 shows the Gram gt11 vector used for preparing the expression library. To make a library of cDNAs, using 2 micrograms of poly A-mRNA isolated from ovarian tissue,
Each cDNA was synthesized. The first-strand cDNA was primed for oligo dT and reverse transcrip
tase). Add reverse transcriptase, 4
After incubation at 3 ° C. for 60 minutes, the reaction is terminated by the addition of EDTA. Samples are extracted using phenol: chloroform: isoamyl alcohol (25: 24: 1). The aqueous phase was removed and the organic phase was 1 mM
Was extracted again with 0.1 M NaCl containing 10 mM Tris-HCl containing EDTA (pH 8.0). After pooling the aqueous phase,
After precipitating the DNA with 2M ammonium acetate, 95%
Ethanol was added. After freezing and thawing, the solution was centrifuged and 25 μl of 10 mM Tris-HCl, 1 mM EDTA (T
Redissolved in E). 7.5M ammonium acetate
Add 10 micrograms, 50 μl of 95% ethanol. RNA-DNA hybrid is E. coli RNAase H
Nicked by Second stran
d) cDNA synthesis was performed with E. coli DNA polymerase. Double strand cDNA is T4D
It was blunt-ended by NA polymerase (blunt-e
nded). The cDNA was methylated at the EcoRI site before ligation to EcoRI linkers. Linked cDN
A was then treated with the EcoRI enzyme, purified by chromatography on BioGel P50 (BioRad) and, based on the method described herein, λgt1 obtained from Strategen.
One arm was ligated. According to this method, about 5 weeks for a 6 week rabbit and 8 month rabbit library.
x10 pieces of plaque can be obtained, 1x10 ⁷ pieces of plaques were obtained for the 12-week rabbit of the library.

When the library was screened, 10
5 × 10 ³ spots were obtained per 0 mm plate. next,
The spots were transferred to nitrocellulose paper impregnated with IPTG, and the probe used was the antiserum prepared in the volume described in Example 2. Because protein sequences are similar in many species, it is necessary to separate protein sequences that express cDNA. For this, polyclonal antibodies against rabbit ZP protein were affinity-purified on a porcine ZP column (ZP protein bound to cyanogen bromide activated sepharose as in Example 2). Antibodies eluted with 0.1 M glycine (pH 2) are able to recognize antigens associated with rabbit and porcine ZP proteins.

Λgt11-S Expressed with ZP Antigen
1, clones of λgt11-P1, λgt11-P2 and λgt11-P3 were subclones. These subclones are cloned in M13 phage using the Sanger dideoxynucleotide chain termination method to form the sequence of the cDNA (Proc. Natl. Acad.
Sci. USA 74: 5463, 1977, Sanger et al.). The sequences obtained using this method for three cDNA clones that express the ZP antigen are shown in FIG.

Using Northern blot analysis, it was demonstrated that the ZP protein RNA recognized by the two cDNA clones (P2 and P3) was present in the ovaries but not in other tissues. Was. FIG. 5 shows one of these analyses, which was performed by hybridizing a labeled probe to an RNA sample using "Northern blot" and "dot blot".
That is, in the case of Northern blot analysis, all RNs
As well as being separated from A ovaries, it was similarly separated from other tissues including liver, kidney, brain and muscle. RNA
Was denatured on a formaldehyde agarose gel and electrophoresed. Next, a biotin membrane (bio
(dyne membrane). The membrane was placed between two paper filters for transfer, air dried and baked at 80 ° C for 2 hours. λ
The unique ZP cDNA inserted into gt11 is EcoRI
Digested by The insert was then resolved from the cloning vector by electrophoresis on 1% low melting point agarose. Under UV light, insert DN
The A molecule was identified with ethidium bromide and scraped from the gel for radiolabelling. Anal. Biochem. 132: 6
(1983), cDNA was labeled by the random oligo-priming method described by Feinberg and Vogelstein. The labeling reaction was performed by adding the denatured cDNA to reagents. The reagents contained ³² P-dCTP, krenow fragment, BSA, mixed primer, and oligolabeling buffer. The labeled DNA was separated from dCTP by chromatography on a Sephadex G-50 column. DNA
-For RNA hybridization, the biosin membrane to which the RNA was transferred was separately placed in a heat-sealable polyethylene bag. Add the prehybridization solution into the bag,
The bag was immersed in a 42 ° C. water bath overnight. Remove the pre-hybridization solution was replaced with hybridization solution containing the DNA probe labeling ³² P, was immersed in overnight in 42 ° C. water bath. Finally, the membrane was washed several times, darkened and exposed to X-ray film. Before developing the film, at 70 ° C
Illuminated for 48 hours.

Example 4 Expression of ZP Protein by Recombinant DNA Recombinant ZP protein (rZP) is expressed in prokaryotic or eukaryotic expression lines. The recombinant λgt11 prepared in Example 3
Phage, λgt11-S1, λgt11-P1, λgt11-
P2 and λgt11-P3 were prepared at 32 ° C. in Luria Broth (LB) medium containing 10 mM MgCl ₂ .
E. coli Y1089 was infected by culturing with E. coli Y1089 for 20 minutes. Colonies were grown at 32 ° C. In order to examine the temperature sensitivity, only one group grew at 42 ° C. It grows at 32 ° C,
Populations that do not develop at 42 ° C are considered lysogenic and make up about 10-70% of the total population. One population of recombinant resogens was isolated and used to inoculate LB media and grown at 32 ° C. When OD600 is about 0.5,
The culture temperature was changed to 42 ° C., and the cells were further cultured for 20 minutes. Transcription of the cloned gene was stimulated by the addition of isopropyl β-D thiogalactopyranoside (IPTG) to a final concentration of 10 mM and incubated at 37 ° C for 60 minutes.
Transformed E. coli can be obtained by centrifugation, resuspended in 50 mM Tris (Ph 7.5) and frozen. Thawing of the cells causes a cytolytic effect, releasing the fusion ZP protein.

A bacteriophage containing λgt11-P1 and λgt11-P3 designed with ZP DNA was 198
On October 8, 2007, deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland, USA. Deposit numbers are 40377 and 4
0378. The deposited bacteriophage is available under U.S. patent laws and regulations. Also available to citizens of countries other than the United States for which a corresponding application has been filed. The fact that the deposited bacteriophage can be obtained does not mean that the present application and the divisional application based on this application are granted the license of the patent granted.

In another embodiment, EMBO 3 (6): 1429 (19
E. coli Y1090 can be transformed using the pEX plasmid disclosed by Stanley and Lugio in (84). The pEX vector expresses the insoluble hybrid protein. Isolation of bacteriophage λ DNA is described in Cold Spring Harbor Laboratory (1982), p.
7 can be performed on recombinant λgt11 phage according to the method described in Molecular Cloning by Maniatis et al. 10 mM MgCl ₂ , 0.2% maltose and 50 μg / ml
(Supplemente) supplemented with Ampicillian
d) E. coli Y1090 grown in LB medium contains recombinant λ
The cells were infected with gt11 phage and cultured at 37 ° C for 20 minutes.
Infected E. coli was placed on LB plates in LB media with 0.7% agarose, MgCl ₂ and ampicillin,
Cultured overnight at 43 ° C. Phage was SM medium (0.02M
Tris -HCl, pH 7.5; 0.01M NaCl; 0.001M MgSO 4 · 7H 2
O) (pH 7.5) and 1% water 0.01% gelatin) and the bacteria are removed by centrifugation at 8,000 xg for 10 minutes at 4 ° C. RNase A and D
NaseI was added to the supernatant containing the phage at a final concentration of 1 μg / ml and incubated at 37 ° C. for 30 minutes. Add an equal volume of SM containing 20% polyethylene glycol and 2M NaCl, incubate the suspension for 1 hour at 0 ° C., 10,000 × g
At 20 ° C. for 20 minutes. The supernatant was removed and the phage-containing pellet was resuspended in 0.5 ml M medium. The solution is 8,000 to remove residues.
xg, centrifuged at 4 ° C for 2 minutes, 5 μl of 10%
SDS and 0.5 M EDTA (pH 8) were added to the supernatant, and cultured at 68 ° C for 15 minutes. As a result, phenol, phenol: chloroform: isoamyl alcohol (25: 24: 1),
And chloroform: isoamyl alcohol (24: 1) was extracted and an aqueous phase could be obtained at each stage. An equal volume of isopropanol was added to the aqueous phase and the solution was stored at -70 C for 20 minutes. After centrifugation at 4 ° C. for 10 minutes, the pellet was washed with 70% EtOH, dried and resuspended in 50 μl of TE. After digestion of EcoRI restriction enzymes, partial samples of the isolated DNA were prepared by standard methods.
After digestion was completed, Tris boronate EDTA loading buffer (TBE) 90 mM Tris borate, 90 mM boric acid, 2 mM EDTA was added at a ratio of 1: 1 and loaded on a 1.4% agarose gel. The gel was run at 100 V until the xylene cyanol front was in the middle of the gel.
n). DNA bands are visually recognized by ultraviolet light, and bands of the expected size are removed. In a TBE Elutrap device, electroeluti
on). The separated DNA fragment is then:
Inserted into pEX plasmid.

The preparation of the plasmid was performed as described in Manitias (1982).
It can be executed based on the method described above. After overnight growth of bacterial clones, 1.5 ml cultures were transferred to Eppendorf tubes (Ep.
pendorf tube) and the supernatant was removed by suction. The pellet was resuspended in 100 μl of ice-cold solution. The solution contains 50 mM glucose, 10 mM EDTA, 25 mM
Tris (pH 8.0) and 4 mg / ml lysozyme are included. After the suspension, the cells were cultured at 22 ° C for 20 minutes, and cooled on ice.
A further 200 μl of 2M NaCl and 1% SDS were added. 5
After culturing on ice for 150 minutes, 150 μl of 3M potassium acetate (pH
4.8) was added. The solution was incubated on ice for 5 minutes, then centrifuged and the supernatant was transferred to another fresh one. Add 600 μl phenol: chloroform: isoamyl alcohol (25: 24: 1), mix the solution and centrifuge to save the supernatant. Next, two volumes
Of cold ethanol is added to the supernatant, the supernatant is mixed and incubated at 22 ° C. for 2 minutes. The supernatant is removed and the pellet is dried by inverting the tube. Pellets 70% Et
Wash with OH, centrifuge and drain again. 20μg / ml
Of DNase-free pancreatic RNase containing 50 μl of TE (p
H 8.0) was added, mixed gently, and aliquots were removed for digestion with EcoRI. Digestion analysis is performed by agarose gel electrophoresis.

The plasmid is linearized with EcoRI (ProMega BioTech) under the conditions specified by the manufacturer. 200 ng of the linearized plasmid DNA was tripled (3-fo
ld) to the molar excess of the isolated cDNA. DNA is EtO
Sediment with H and resuspend in 8 μl TE (pH 8.0). 1 μl of 10 × ligated 66 mM Tris-Cl (pH 7.6),
A buffer of 6.6 mM MgCl2, 10 mM dithiotitre, 66 mM ATP was added, followed by T4 DNA ligase (ligas
e) was added in 10 units. The mixture is incubated at 12 ° C. for 8 hours. One microliter aliquots are analyzed by agarose gel and ligated. Bacterial transformation is performed with 2.4 μl by CaCl ₂ technology.

The population carrying the recombinant plasmid can be identified by screening the bacteria with a polyclonal antiserum. The transformed cells are grown at 30 ° C. for 20 hours. Nitrocellulose is formed thereon, the filter is removed and the population is placed side by side under two layers of a 3MM paper filter presoaked in LB media containing 100 μg Amp / ml. By culturing at 42 ° C. for 2 hours, transcription of the hybrid protein starts. Filter is T
Wash 3 times every 30 minutes in BS and occlude overnight in TBS supplemented with 2% dry milk. The filters are then probed with affinity-cleared antibodies. Positive population (pos
(colonic colonies) at 32 ° C. Expression of ZP protein is brought about by changing the temperature to 42 ° C. and culturing for 2 hours. At this time, the recombinant ZP
The protein is about 25% of the total SDS extracted protein. Expressed hybridized ZP protein is insoluble and can be easily cleaned up by conventional chromatographic methods.

Alternatively, the recombinant ZP DNA can be inserted into yeast. By using a methyl-atrophic yeast such as Pichia pastoris, a genetically designed protein can be expressed efficiently and on a large scale (Chemical Week, McGraw-Hill, (198
6) M. Bluestone and P. Savage). These yeasts use methanol as a brew base, and the yeast is metabolized into formaldehyde with the aid of alcohol oxidase. The second enzyme then converts formaldehyde to dihydroxyacetate and performs the next stage of yeast cell synthesis. cDNA is
It is inserted at the position of the gene that oxidizes alcohol. Z
Recombinant DNA from the P protein is replaced at the site where the alcohol oxidase gene is normally present. Since this site is altered in yeast, it responds to methanol. Therefore, by simply switching to ethanol or carbohydrate, the inserted gene can be expressed or not expressed. Two methanol-regulated lacZ (lacZ) genes can be expressed in Pichia pastoris (Nucleic Acids
Research 15 (9): 3859 (1987), Chop et al.).

Example 5 Purification of Recombinant Zona Zona Protein The recombination of ZP protein is expressed as described in Example 4. E. coli Y1089 was infected with λgt11 recombinant lysogen. Escherichia coli is supplemented with 0.2% maltose at 32 ° C to an optimum density (600 nm) of 0.5 LB.
Developed in the medium. Next, the culture temperature was changed to 42 ° C., and the culture was further performed at that temperature for 20 minutes. At the end of this culture, I
PTG was added to reduce the rack repressor. As a result, the gene in the rack Z direction including the recombinant is expressed. The cells are cultured at 37-38 ° C for 60 minutes,
Freeze in liquid nitrogen and thaw to lyse the cells. Lysate was collected in 0.1 M Tris buffer (pH 7.5) and passed through a Sephacel column. Voids containing proteins with molecular weights greater than 100 Kd (voi
Those with a volume of d) were collected. The expressed protein is in this case fused to the β-galactosidase protein, which may be a rapid method of partially cleaning this protein. This protein fraction is analyzed by SDS-PAGE to detect the expressed antigen by immunoblotting.
FIG. 6 shows an SDS-PAGE immunoblot of the ZP antigen fusion protein expressed by the P1 clone. Nitrocellulose transfer was probed with rabbit anti-HSPZ. Many low molecular weight bacterial proteins are recognized in all bacteria, but for P1 insertion, two lanes are
3 shows the expression of a fusion protein specific to P.

The protein is separated from the pEX plasmid by extracting the protein from bacteria. The hybridized protein of the recombinant clo-β-galactosidase is about the same as the protein extracted with sodium dodecyl sulfate.
Because it is 25%, it can be deposited by Sephacel and ion exchange columns or by electrophoresis.
For this reason, the protein can be cleaned so as to obtain sufficient cleanliness necessary for immunization of the animal. Recombinant proteins isolated from yeast culture media can also be cleaned by these methods.

Example 6 Zona immunization It was found that active immunization or autoimmunity of zona pellucida proteins was effective in suppressing fertilization in animals. By imparting active immunity to rabbits, the fertilization function in ovarian follicles can be impaired, and steroid hormone production can be stopped (see FIG. 7).

ZP protein (300 μg / 0.5 ml; 0.1 M P
BS) with 0.5 ml of Freund's complete adjuvant (CF
Emulsify in A). Half of the dose was administered to many sites in the skin, and the other half was 0.5 ml emulsified with 0.5 ml PBS.
Injected with emulsion of CFA. With this injection, the animals are boosted after 4 weeks. The immunity is otherwise identical when compared to the first, except that the first uses incomplete Freund's adjuvant. To be able to assess ovarian function, animals underwent hCG treatment to determine serum progesterone levels. Some animals in each group were sacrificed at least 12 hours after hCG administration, ovaries were weighed and ovulation sites were examined. On the other hand, hCG
Prior to, animals were given porcine FSH (Reheis Chemical Co., Phoenix, AZ; 0.5 mg / animal, twice daily, for 3 days) and hyperovulation. The results of these studies revealed that the causes of changes in ovarian function were lack of follicular cell differentiation and steroid production (see FIG. 7). The level of serum progesterone in rabbits immunized with ZP in pigs was determined. The response of the animals given 50 IU hCG showed progesterone levels of RIA.
Determined by The serum samples tested were collected periodically for 21 days. After this hCG treatment, pseudopregnancy usually occurs. Each histogram represents the mean ± S.E. of serum progesterone concentration (nanograms of progesterone per ml of serum).
Represents D.

Panels A and D show the former as a control.
Animals (adjuvant only), the latter show the progesterone profile before immunization, respectively, of experimental animals (immunized with ZP). B and E show the state 20 weeks after the first immunization treatment. C and F
Shows the progesterone profile of non-enhanced animals, which shows FS 28 weeks after the first immunization.
After H / hCG treatment. The use of monoclonal antibodies R5 and R7 for passive immunization reduced the fertilization rate of rabbits. Monoclonal antibodies designated by PSI are made against silver-stained porcine ZP protein. The protein has been purified by 2D-PAGE. This monoclonal antibody inhibits porcine serum from being fixed to pig ZP in vitro (see FIG. 8).

Example 7 Preparation of Anti-Zona pellucida Monoclonal Antibody A hybrid cell line producing an anti-Zona pellucida monoclonal antibody was prepared. BALB / c murine myeloma cells were prepared by priming BALB / c with zona pellucida protein. It fuses with spleen cells of Mice. In this way, the myeloma cells and the anti-zona pellucida antibody-producing cells can utilize other sources, such as humans, dogs, or cats.

A. Preparation of spleen cells for fusion Zona pellucida is ovarian or highly degradable 2D-PA
The separation by GE is to be cleaned as all heat solubilized matrices. Zona pellucida antigens
In 2D-PAGE analysis, it was approximately 95% pure (ZP solubilized thermally), and in silver staining analysis, it was 100% pure. Zona pellucida antigen, Freund's complete adjuvant about 30
μg emulsified in BALB / c or C
It is used to immunize 57B46 male mice by subcutaneous administration. Mice were re-immunized two weeks later with an additional 30 μg of protein in incomplete adjuvant for another 6 weeks.
After a week, 20-40 μg of protein was administered intravenously. Two to four days later, Meis died and a suspension of spleen cells was made based on the method disclosed by Gefter et al. In Somatic Cell Genetics 3: 231, 1977. Lysis red blood cells, NH
_The cells were cultured in ₄ Cl (0.83%) at 40 ° C. for 15 minutes. The resulting suspension is washed through heat-inactive calf serum by centrifugal action (800 xg) and then the protein-free medium (R
Centrifugal force was applied in RMI 1640, buffered with 7.5 nM HEPES, pH 7.2).

B. Preparation of spleen for fusion Spleen cells were obtained from the P3U1 line and were obtained from Curr. Top.
81: 1-7 (1978), lack of HPRT (EC 2.4.2.8) as described by Yelton et al. Transfer these cells to Eagle's minimum essential medium (minimum essen
tial medium, MEM). This medium contains 10% calf serum and 15% horse serum. Myeloma cell growth is controlled by hypoxanthine-aminopeterin-thymidine (HA).
T) Suppressed by media selection.

C. Hybrid Production Hybrid production consists of 107 BALB / c myeloma cells,
It was performed by mixing 10 ⁸ spleen cells obtained from BALB / c or C57B1 / 6 MICE given immunity zona. The mixture of cells is centrifuged at 800xg,
Cells were resuspended in a 50% solution of polyethylene glycol (PEG 1000) diluted in serum free minimum essential medium (MEM) based on the method of Gefter et al. (1977). The resulting hybridoma cells were isolated from hypoxanthine by limiting the diluent, as described by Gulfre and Milstein in Meth. Enzymol. 73: 3, 1975.
Clones were formed in aminopeterin-thymidine (HAT) medium. Two hybridoma cell lines R5 and PS
I was selected. The reason for this is that cell line R5 makes antibodies that recognize the protein portion of the ZP antigen in many animals. PSI also recognizes carbohydrate determinants of many species of ZP, as this antibody suppresses sperm binding to the surface of ZP (see FIG. 8).

The hybridoma cell line designated R5 and PSI was deposited on October 8, 1987 with the American Type Culture Collection (ATCC), Rockville, Maryland, USA. The deposit numbers are 40377 and 40378. The deposited bacteriophage is available under U.S. patent laws and regulations. Also available to citizens of countries other than the United States for which a corresponding application has been filed. The fact that the deposited bacteriophage can be obtained does not mean that the present application and the divisional application based on this application are granted the license of the patent granted.

D. Test for clones producing anti-Zona pellucida 96 for microtiter of Limbro (Linbro, Flow Lab)
Individual welled plates were coated with 50-100 μg of rabbit or porcine ZP protein and incubated at 20 ° C. overnight. The cavity is 0.1% Tris (pH 7.5) -1% non-idet containing 5% Carnation Instant Milk and 0.085% sodium azide
After washing with (nonidet) P-40, 0.05 ml of culture supernatant was added and cultured at 40 ° C. The supernatant was removed after washing three times with RIA buffer and used for hybridoma screening.
Antibodies were detected using a kit (Bethesda Research Labs). Non-specific binding controls do not include either the second antibody or the culture supernatant.

The hybridoma cells called R2, R5, R7 and PSI were treated with pristane (pristane-tr
Eated) grew as a form of ascites by intraperitoneal injection into mice (Meth. Enzymol. 73 (Part B) 1 (1981), Gulfre and Milstein). Ascites fluid was collected and used as a source of monoclonal antibodies for immunoblots and bioassays.

The present invention has been described in detail. It will be apparent to one skilled in the art that modifications may be made without departing from the spirit or scope of the invention as set forth in the appended claims.

[Brief description of the drawings]

FIG. 1 is a diagram showing antibody-recognizing ZP antigens used for screening libraries by 2D-PAGE gel immunoblotting.

FIG. 2 shows N-terminal amino acid sequences of porcine ZP protein and rabbit ZP protein.

FIG. 3 shows a cleavage map of the restriction endonuclease of the λgt11 vector, which is used to develop an expression library for cloning the cDNA of ZP.

FIG. 4 shows the DNA sequences of three clones that express ZP antigen.

FIG. 5 shows Northern blot analysis showing that ZP RNA is present in the ovaries but not in other tissues.

FIG. 6 shows SDS- of λgt11 DNA from clone P1.
It is a figure which shows a PAGE immunoblot.

FIG. 7 shows the results of active immunization of rabbits for ovarian function.

FIG. 8 shows that monoclonal antibodies suppress serum binding of sperm to ZP.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 5/10 C12P 21/00 C 15/02 21/08 C12P 21/00 C07K 16/18 21/08 C12N 5/00 B / / A61K 38/00 15/00 C C07K 16/18 A61K 37/02 (C12N 1/19 C12R 1:84) (C12N 1/21 C12R 1:19) (C12P 21/00 C12R 1:19) (C12P 21 / 08 C12R 1:91)

Claims (29)

[Claims] 1. The P2 sequence, P2 sequence shown in FIG. 4 by the addition, deletion or substitution of one or more amino acids.
A substantially pure DNA sequence encoding a zona pellucida polypeptide comprising an amino acid sequence different from the amino acid sequence encoded by the 3272 base sequence or the P3 484 base sequence, wherein the encoded polypeptide is a P2 sequence. , P3 272
It contains an antigenic determinant specific to the amino acid sequence encoded by the base sequence or the P3484 base sequence. 2. A DNA comprising an expression carrier comprising the DNA of claim 1 under control of a sequence for expression of the encoded polypeptide. 3. A host cell transformed with the DNA of claim 1 or 2. 4. The host cell according to claim 3, wherein the host cell is a prokaryote. 5. The host cell E. coli (E. Coli.) In a fourth aspect of the host cell. 6. The host cell according to claim 3, wherein the host cell is yeast. 7. The host cell according to claim 3, wherein the host cell is a mammalian cell.
Host cells. 8. Use of the DNA of claim 1 or 4 for transforming a host cell. 9. Use of a host cell according to any of claims 3 to 7 for the production of an encoded polypeptide. 10. A method of producing a polypeptide, comprising: a) transforming a host cell with the DNA of claim 1 or 2; and b) producing the encoded polypeptide.
C) recovering the expressed polypeptide from the host cell;
A method for producing a polypeptide comprising: 11. The host cell of claim 10, wherein the host cell is a prokaryote. 12. A host cell E. coli (E. Coli.) A host cell of claim 11. 13. The host cell of claim 10, wherein the host cell is yeast. 14. The host cell is a mammalian cell.
10 host cells. 15. The method according to claim 10, comprising producing a composition comprising the polypeptide and a pharmaceutically suitable carrier.
14. The method according to any one of to 14. 16. The method of claim 15, wherein the composition comprises an adjuvant. 17. The method of claim 15, further comprising administering the composition to a mammal other than a human to cause contraception.
16 ways. 18. The method of claim 17, wherein said administering is by infusion. 19. The method according to claim 17, wherein the non-human mammal is a cow, pig, cat, dog or rabbit. 20. A pharmaceutical composition comprising: a) a pharmaceutically suitable carrier; b) recombinant expression comprising encoding the DNA in a transformed prokaryotic or eukaryotic host cell. A substantially pure, non-naturally occurring polypeptide produced in an amount effective to produce immunity, wherein said polypeptide has no naturally occurring sugar chains, and said polypeptide comprises:
The polypeptide comprises an amino acid sequence that differs from the amino acid sequence encoded by the P2, P3272 or P3484 base sequence shown in FIG. 4 by the addition, deletion or substitution of one or more amino acids. , P2 sequence, P
A pharmaceutical composition comprising an antigenic determinant specific for the amino acid sequence encoded by the 3272 base sequence or the P3484 base sequence. 21. The pharmaceutical composition according to claim 20, wherein the carrier comprises an adjuvant. 22. A method comprising contraception in a mammal, comprising administering the composition of claim 20 or 21 to a non-human mammal to cause contraception. 23. The method of claim 22, wherein administering is by infusion. 24. The method according to claim 22, wherein the mammal is a cow, pig, cat, dog or rabbit. 25. The method of claim 22, wherein the mammal is a human. 26. A claim effective in generating immunity.
A method for producing in a mammal an antibody directed against the zona pellucida antigen encoded by DNA shown as P2 or P3 shown in FIG. 4 by administering the pharmaceutical composition of 20 or 21 to the mammal. 27. The method of claim 26, wherein administration is by infusion. 28. The method according to claim 26, wherein the mammal is a cow, pig, cat, dog or rabbit. 29. The method of claim 26 or 27, wherein the mammal is a human.