CN1158623A - New immunocotraceptive peptides - Google Patents
New immunocotraceptive peptides Download PDFInfo
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- CN1158623A CN1158623A CN95195212.9A CN95195212A CN1158623A CN 1158623 A CN1158623 A CN 1158623A CN 95195212 A CN95195212 A CN 95195212A CN 1158623 A CN1158623 A CN 1158623A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The present invention relates to a peptide capable of inducing an immune response against the Zona Pellucida protein ZP3, said peptide having an amino acid sequence of 8-50 amino acid residues and comprising at least the amino acid sequence PLWLLQ or analogues thereof. More specifically, the peptides according to the invention comprise at least the amino acid sequence QPLWLLQG. Further embodiments of the invention relate to antibodies raised against the peptides according to the invention, contraceptive vaccines comprising said peptides or said antibodies, as well as a test kit for the detection of autoimmune antibodies against ZP3 in a test sample.
Description
The present invention relates to bring out the peptide of the immunne response of anti-zona pellucida protein ZP3, with this peptide produce can with the purposes of the immunoreactive antibody of its generation, monoclonal antibody, can produce the hybridoma cell line of described antibody, the purposes that described peptide or described antibody is used to prepare pregnancy vaccine, the pregnancy vaccine that contains described peptide or antibody, described peptide is used for the purposes of the autoimmune antibody of the anti-ZP3 of test sample, contains the diagnostic kit of described antibody and be used for the test kit of the autoimmune antibody of the anti-ZP3 of test sample.
Zona pellucida (ZP) is the outer glycoprotein matrixs of the compound born of the same parents around the mammal ovocyte.This matrix formed at oocyte maturation initial stage and follicular development initial stage, and it comprises the albumen of three kinds of high glycosylations, is called as ZP1, ZP2 or ZP3.Described ZP plays an important role in fertilization process, because interact being first between the Mammals gamete caused by combining of the special acceptor on sperm and the ZP.In mouse, ZP3 glycoprotein has been proved to be the sperm-receptor (it is summarized referring to Wasserman, Development 108:1-17,1990) that is positioned at the complementary molecule in the sperm surface film.The above-mentioned ligand function of ZP3 can cause a kind of process that is called as acrosomal reaction, and the result of this reaction can discharge proteolytic enzyme, makes described sperm penetrate zona pellucida and makes oocyte fertilization by merging with the plasma membrane of ovocyte.
ZP3 glycoprotein in essence-ovum interacts vital role and the fact of ovocyte specifically expressing point out us, ZP albumen can be used as the ideal target antigen, is used to develop pregnancy vaccine (for example, Gwatkin etc., Fert.Ster.28:871-877,1977; Paterson and Aitken, Gurr.Opinion in Immunol., 2:743-747,1990).Confirm all that in a lot of researchs the zona pellucida protein of inoculating pig to Mammals can produce anti-used antigenic high antibody titer, and follows sterile.By in vitro study, also can obtain the result of the contraceptive effect of indication ZP3 immunity.Confirmed already that anti-ZP3 specific antibody can suppress microgamete with the combination of salt storage human oocyte (van Duin etc., Hum.Reprod.9 (Suppl.4): 41,1994), but also can destroy (Henderson etc., Gam.Res., 1988) in vitro fertilization of human body.
With natural ZP3 albumen is target antigen, and the vaccine for preparing based on ZP3 by engineering method is infeasible, because this biomaterial is very limited.Clone to coding ZP protein gene has solved the problems referred to above recently, produces reorganization ZP3 albumen or its segmental possibility thereby provide.The clone of described mouse ZP3 DNA and evaluation have constituted important step (Ringuette etc., Proc.Natl.Acad.Sci.USA 83:4341-4345,1986 of this purpose of marching toward; Rin-guette etc., Dev.Biol., 127:287-295,1988).The clone and the relevant proteic one-level aminoacid sequence of ZP3 of various Mammals ZP3 genes have been disclosed, as people ZP3 (WO 90/15624), hamster ZP3 (Kinloch etc., Dev.Biol., 142:414-421,1990), marmoset ZP3 (WO 94/10304), pig ZP3 (WO 93/14786), cat ZP3 (JP-A-06014784; WO 94,11019), dog ZP3 (JP-A-05336974; WO 94,11019), rabbit ZP3 (WO 94/11019), ox ZP3 (WO 94/11019) and macaque ZP3 (WO 94/11019).All illustrated so far Mammals ZP3 aminoacid sequences all show very high evolution conservative, and amino acid whose homology is in 65～90% scope.The conservative property of this proteic N-end of different mammalian species is relatively poor, and this shows that the specific specificity of proteic this part of ZP3 in the Mammals fertilization process has latent effect.
A lot of Mammalss with complete ZP3 protein immunization in most of the cases can both produce high anti-ZP3 and tire, and follow sterile.But, about also having mentioned the result of following unanimity in the report of above-mentioned research: the ovarian function that shows as the disorderly menstrual cycle is not normal, ovariopathy is of science and the forfeiture of the ovarian follicle of each etap, the forfeiture that comprises primordial follicle (for example, Skinner etc., Endocrinology, 115:2418-2432,1984; Upadhyay etc., Biol.Reprod.41:665-673,1989; Sehgal etc., Pathology 21:105-110,1989; Dunbar etc., Fert.Ster.52:311-318,1989; Jones etc., J.Reprod.Fert.95:513～525,1992; Mahi-Brown etc., J.Reprod.Immunol.21:29-46,1992; Paterson etc., Biol.Reprod.46:523-534,1992).This malfunction can cause permanent sterile.Especially concerning human body, this is a kind of side effect of very cakes with moulded designs, because only need be used for the control fertilization based on the immunological contraception effect of the vaccine of ZP3, therefore, the sterility of being brought out preferably can automatically or by the mode of operation recover normal.Therefore, complete ZP3 is not suitable for the safe contraceptive bian of exploitation.
ZP3 plays a part antigenic determinant, and it can bring out by T cell or B cell-mediated immune responses.Described antigenic determinant can be called t cell epitope or B cell epitope according to the bring out type of replying.With the mouse is model, has confirmed with viewed ovariopathy Neo-Confucianism behind the ZP protein immunization it is a kind of by the cell-mediated phenomenon of T.After carrying out immunity with a kind of 8 amino acid whose little t cell epitopes (the amino acid 330-337 that is equivalent to mouse ZP3), can bring out the ovariopathy Neo-Confucianism of mouse, be specially ovaritis.The T cell transfer of described animal can be caused the ovary damage equally in non-immune animal body, this result has supported that effectively t cell epitope is the view (Rhim etc. that cause the reason of ovary damage after the ZP3 immunity, J.Clin.Invest.89:28-35,1992; Luo etc., J.Clin.Invest.92:2117-2123,1993).
Therefore, in order suitably to prepare the pregnancy vaccine based on ZP3, press for the proteic fragment of ZP3, it does not contain the pathogenic T cell epitope of ZP3.An object of the present invention is to provide a kind of peptide, it is equivalent to the proteic single antigenic determinant of ZP3, and therefore, this antigenic determinant can not brought out by the cell-mediated pathogenic immunity of T and should be signed.
The invention provides a kind of like this peptide.Peptide of the present invention has the aminoacid sequence of being made up of 8-50 amino acid, and, comprise aminoacid sequence PLWLLQ (SEQ ID NO:1) or its analogue at least.
More particularly, peptide of the present invention comprises aminoacid sequence QPLWLLQG (SEQ IDNO:2) or its analogue.
The flank site of aminoacid sequence PLWLLQ on the peptide of the present invention (SEQ ID NO:1), particularly the flank site of sequence QPLWLLQG (SEQ ID NO:2) is equivalent to the natural flank site of the amino acid 24-29 of people ZP3 aminoacid sequence, particularly the natural flank site of amino acid 23-30 perhaps is equivalent to the non-natural flank site of being made up of any aminoacid sequence at random.
Peptide of the present invention has the aminoacid sequence of being made up of 8～50 amino-acid residues, and 8-35 better, and 8～25 better.Peptide with aminoacid sequence of being made up of 8～15 amino-acid residues is particularly desirable.Special recommendation has the peptide of the aminoacid sequence of being made up of 8 or 12 amino-acid residues.
Scope of the present invention also comprises the polymer of being made up of peptide of the present invention, as binary or trisome.This polymer can provide the multiple special aminoacid sequence of multiple PLWLLQ and/or QPLWLLQG, causes immunne response by it.
In this article, analogue is meant which has the peptide that replaces or replace, insert or lack in the polymer of aminoacid sequence PLWLLQ or QPLWLLQG (SEQ ID NO:1 and 2) or described sequence, its prerequisite is, these analogues can with by preserving number be the monoclonal antibody generation immune response that 94032402 hybridoma cell line produces, this clone has been transferred to European zooblast preservation center (herein hereinafter to be referred as ECACC) preservation on March 24th, 1994, this center is located at Port Down, Salisbury (UK).Described replacement or displacement are not must be as the described conservative substitution of M.O.Dayhoff (Atlas of ProteinStructure, Vol.5, Suppl.3 Natl.Biomedical Research Foundation, 1978), and can be non-conservative amino-acid substitution, this non-conservative substitution can cause occurring the mimicry (mimitope) of aminoacid sequence PLWLLQ or QPLWLLQG (SEQ ID NO:1 or 2).
The said mimicry of this paper is meant a kind of aminoacid sequence, it is different from sequence shown in SEQ ID NO:1 and 2, but it can bring out other antibody, the epi-position of this antibody capable identification with by the ECACC preservation, preserving number is that the epi-position discerned of 94032402 the monoclonal anti physical efficiency that hybridoma cell line produced is identical.The method of identifying this mimicry is already by disclosure (Proc.Natl.Acad.Sci.USA 81:3998-4002 such as Geysen; 1984; J.Immunol.Methods 134:23-33; 1987).This technology is widely known by the people in this area, and the test kit that has reagent and a material also can be obtained by commercial sources.
For example, at least the peptide that comprises aminoacid sequence PLWFWQ (SEQ ID NO:3) or PMWTLQ (SEQ ID NO:4) with aminoacid sequence of forming by 8～50 amino-acid residues, be can with by the ECACC preservation, preserving number is that immunoreactive analogue takes place for 94032402 the antibody that hybridoma cell line produced.
Appropriate peptide of the present invention is for having peptide or its mixture of aminoacid sequence QPLWLLQG (SEQ ID NO:2), PQPLWLLQ (SEQ ID NO:5), PLWLLQGG (SEQ IDNO:6), LCYPQPLWLLQGGASHPETS (SEQ ID NO:7) or ADGAPMWTLQGAAGA (SEQ ID NO:8).
A kind of preferred peptide that is used to develop pregnancy vaccine is the peptide with aminoacid sequence QPLWLLQG (SEQ ID NO:2).
With peptide vaccination of the present invention and after producing higher antibody titer, do not observe the sign that ovarian dysfunction or other demonstration ovary sustain damage.Peptide of the present invention can not bring out the cell-mediated pathogenic immunne response by T, thereby be specially adapted to develop can be at the pregnancy vaccine of ZP3 immunity.
Millar etc. have disclosed the peptide (Science 246:935-938) that can not bring out in by the cell-mediated pathogenic immunne response of T previously.Millar confirms, is equivalent to amino acid 336-342 small-sized antigenic determinant of mouse ZP3 aminoacid sequence, after female mice is carried out active immunity, can produce contraceptive effect, but can not bring out ovariopathy Neo-Confucianism.But, this peptide is different from peptide of the present invention fully.Peptide of the present invention is included in aminoacid sequence listed in SEQ ID NO:1 or 2 or its analogue, and this sequence is equivalent to the amino acid 24-29 in the people ZP3 aminoacid sequence, particularly amino acid 24-30.Millar does not propose or hints peptide of the present invention, or this peptide can not bring out the fact by the cell-mediated pathogenic immunne response of T.
Peptide of the present invention is highly suitable for developing a kind of pregnancy vaccine that can be used for ZP3 is carried out active immunity.A kind of one or more peptides of the present invention that are used for the pregnancy vaccine that ZP3 carries out active immunity is comprised effective dose, and a kind of carrier that can be medicinal.Take described pregnancy vaccine for female mammal, especially woman, can bring out at the immunity of the corresponding antigens determinant on the ovocyte ZP3 albumen and should sign, but can be at all other antigenic determinants on the ZP3.Therefore, and compare, adopt the pregnancy vaccine that contains peptide of the present invention can obtain more low-keyed immunne response based on the vaccine of complete ZP3.The antibody that is produced can suppress combining of spermoblast and ovocyte, thereby causes sterile.
In addition, peptide of the present invention also can be used for developing a kind of pregnancy vaccine that ZP3 is carried out passive immunization.A kind of antibody that is used for the vaccine that ZP3 carries out passive immunization is contained one or more anti-peptides of the present invention of effective dose, and a kind of carrier that can be medicinal.
Can be highly suitable for ZP3 is carried out the pregnancy vaccine of passive immunization with the antibody of aminoacid sequence SEQ ID NO:1 and/or the anti-peptide of the present invention of the immunoreactive energy of 2 generations.
It is desirable to, the antibody of described anti-peptide of the present invention is monoclonal antibody.
A kind of preferred antibody be by the ECACC preservation, preserving number is 9432402 the monoclonal antibody that hybridoma cell line produced.Better monoclonal antibody is people or humanized monoclonal antibody, and it is used to develop the pregnancy vaccine that ZP3 is carried out passive immunization.
The antibody of anti-peptide of the present invention and the clone that can produce this antibody also fall within the scope of the invention.
Give female mammal, particularly the woman takes this pregnancy vaccine, can make described female generation that the antibody colony of immunoreactive homogeneous can take place with aminoacid sequence shown in SEQ ID NO:1 and/or 2, this antibody can also with the amino acid 23-30 by the aminoacid sequence of ZP3, the particularly corresponding antigen determinant generation immune response of amino acid 24-29 representative, but this antibody can not react with other antigenic determinant on the ZP3.Corresponding antigens determinant on the ZP3 of described antibody and ovocyte combines, and can suppress combining of spermoblast and described ovocyte, thereby cause sterile.
In another embodiment of the invention, peptide of the present invention is applicable in the test kit of detection at the autoimmune antibody of amino acid 24-29, the especially 23-30 of the aminoacid sequence of ZP3.In order to detect this autoimmune antibody, in the study subject body, obtain serum sample, and contact with one or more peptides of the present invention, and selectively with a kind of diagnostic reagent contact that contains antibody of the present invention, particularly monoclonal antibody of the present invention.If there is autoimmune antibody, it will react with described peptide.The reaction that might take place comprises agglutination reaction, competing reaction or inhibited reaction.Can realize detection by mode with suitable detection reagent mark peptide of the present invention or antibody to described reaction, actually or mark peptide traget antibody, this will depend on the type that is reacted.
For example, in order to carry out inhibited reaction so that detect above-mentioned autoimmune antibody in the sample, adoptable test kit contains one or more peptides of the present invention that are coated on a kind of solid support, and the monoclonal antibody of the present invention or its segmental diagnostic reagent that contain underlined mistake.Peptide on this reagent and the described solid support combine the competition that can be subjected to the autoimmune antibody in the sample.
For instance, operable support comprises: the inwall of little prospect hole or sample pool, test tube or kapillary, film, filter membrane, test silver or the particle surface such as latex particle, red corpuscle, dye sols, metal-sol or sol particles shape metallic compound, the carrier proteins such as bovine serum albumin (BSA) or keyhole alkalemia azurin (KLH).
The detection reagent that can be used for the reagent antibodies of mark peptide of the present invention or described diagnostic reagent comprises radio isotope, fluorescent chemicals, enzyme, dye sols, metal-sol or is metallic compound or other colloidal sol of sol particles shape.
The test kit of the diagnostic reagent that contains antibody of the present invention, particularly monoclonal antibody of the present invention that comprises one or more peptides of the present invention and used selectively can be used to diagnose the undesirable sterility that autoimmune antibody caused by at ZP3.
In addition, also described test kit can be used to monitor the antibody horizontal of the female mammal of handling with vaccine of the present invention.
Availablely numerously become known for a kind of in the peptide synthetic organic chemistry method or utilize recombinant DNA technology to prepare peptide of the present invention.
It is generally acknowledged that be used for peptide synthetic organic chemistry method and comprise by condensation reaction and connect needed amino acid, condensation reaction is carried out in the homogeneous phase mode, or by means of so-called solid phase.
Described condensation reaction can be carried out as follows:
A) having under the condition of condensing agent, a kind of another kind of compound (amino acid, peptide) condensation with compound (amino acid, peptide) with other reactive group of other reactive group of free carboxyl and protection with free amino and protection;
B) with a kind of another kind of compound (amino acid, peptide) condensation with compound (amino acid, peptide) with other reactive group of other reactive group of activatory carboxyl and free or protection with free amino and free or protection.
Can activate described carboxyl in the following way, particularly including carboxyl being transformed into a kind of acyl halide, trinitride, acid anhydrides, imidazoles thing (imidazolide) or active ester, as N-hydroxyl-succinimide, N-hydroxyl-benzotriazole or to the nitroso-group phenyl ester.
The modal method of carrying out above-mentioned condensation reaction is: the method for carbodiimide method, trinitride method, mixed anhydride method and use active ester, these methods are disclosed in the following document: ThePeptides, Analysis, Synthesis, Biology Vol.1-3 (author: Gross, E. and Meienhofer, J.) 1979,1980,1981 (Academic Press, Inc.).
For instance, the method for utilizing described " solid phase " to prepare peptide of the present invention is disclosed among J.Amer.Chem.Soc.85:2149 (1963) and the Int.J.Peptide Protein Res.35:160-214 (1990).The amino acid whose connection of peptide to be prepared is arranged, often start from C-terminal one side.This method needs solid phase, and the base that responds on this solid phase maybe can be introduced reactive group.For example, described solid phase can be benzene and the multipolymer with Vinylstyrene of reactive chlorine methyl, or gives its active polymerization solid phase by methylol or amine functional group.
For example, the solid phase of a particularly suitable by Wang (1974) disclosed (J.Am.Chem.Soc 95:1328) to alkoxyl group benzylalcohol resin (4-methylol-phenoxy group-methyl-co polystyrene-% divinylbenzene resin).After synthetic, can be on the described solid phase under the cracking at following institute's synthetic peptide of mild conditions.
After synthetic required aminoacid sequence, this peptide is taken off from described resin, pass through subsequently, for example trifluoromethanesulfonic acid or methylsulfonic acid are dissolved in it in trifluoroacetic acid.Also can be by the transesterification lower alcohol, especially methyl alcohol or ethanol are removed peptide from described carrier, in this case, have directly formed the alkyl ester of this peptide.Similarly, can produce the acid amides of peptide of the present invention with the ammonia cracking.
As mentioned above, the reactive group that does not participate in condensation reaction is effectively protected by protecting group, can remove protecting group once more easily by hydrolytic action or reductive action that acid, alkali carry out.Therefore, by to being connected methyl alcohol on the solid support, ethanol, the trimethyl carbinol, benzylalcohol or the nitrobenzyl alkohol and amine being carried out esterification, can realize effective protection to carboxyl.
Can effectively protect amino group to have: ethoxy carbonyl, benzyloxycarbonyl, tert-butoxycarbonyl (t-boc) or to methoxyl group-benzyloxycarbonyl or by sulfonic acid deutero-acidic group; as benzenesulfonyl or p-toluenesulfonyl; but; also can adopt other group; as replacing or non-substituted aryl or aralkyl; for example benzyl and trityl group, or such as ortho-nitrophenyl base-sulfur phenenyl and 2-benzoyl-1-methyl-vinyl.A kind of amino protecting group of particularly suitable is, for example, and the quick property of alkali 9-fluorenyl-methoxycarbonyl (Fmoc) (Carpino and Han, J.Amer.Chem.Soc.92:5748,1970).
More available protecting groups can be found in following document: The Peptides, Analysis, Synthesis, Biology, Vol.1-9 (author: Gross, Udenfriend and Meienhofer) 1979-1987 (Academic Press, Inc.).
Also must the protection lysine amino, and arginic guanidine radicals protected also to be needed.The common protecting group that is used for this purpose has: be used for the Boc-base of Methionin and be used for arginic Pmc-or Pms-or Mbs-base or Mtr-base.
Described protecting group can be removed with various ordinary method cracking, and method therefor depends on the character of special groups, for example, with trifluoroacetic acid or by slight reductive action, as with hydrogen and catalyzer, reduces or reduces with HBr in Glacial acetic acid as palladium.
As indicated above, can prepare peptide of the present invention by the recombinant DNA technology of standard equally.For this reason, the polymeric nucleotide sequence of code book invention peptide or described peptide be inserted a kind of expression vector.Suitable expression vector comprises: plasmid, clay, virus and YAC ' s (yeast artificial chromosome), above carrier comprise and duplicate and express necessary control fragment.Described expression vector can be expressed in host cell.Proper host cell comprises, for example bacterium, yeast cell and mammalian cell.This technology is well-known (Sambrooke etc., Molecular Cloning:a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1989) in the art.
The available standards technology prepares antibody of the present invention.With the method for peptide immune animal such as mouse, and the method for selecting to produce the hybridoma of immunogen monoclonal antibody specific is widely known by the people in the art (for example, referring to the Current Protocols in Immunology.1992 of works such as Coligan; Kohler and Milstein, Nature 256:495-497,1975; Steenbakkers etc., Mol.Biol.Rep.19:125-134,1994).In brief, use and immunogenic carrier albumen the selected animal of peptide multiple injection of puting together as keyhole hemocyanin (KLH).The available antigenic titer plate that is coated with care is to some extent tested the immune response of immunized animal easily in serum, and hybridoma to be electricity by myeloma cell and mouse B cell merge produces, in the substratum that contains the selective agent that is suitable for this purpose, select subsequently.
The method that is used to produce Humanized monoclonal antibodies can comprise gene engineering, and this technology can be used for the structure again or the humanization of described antibody.For example, the complementarity decision fragment that has the mouse monoclonal antibody binding site (CDR ' S) is inserted people's antibody framework region, can produce people's antibody that wherein CDR fragment derives from murine antibody.This " CDR-grafting " method successfully is applied to a lot of therapeutic purpose, for example, and the antibody of anti-interleukin-22 acceptor (Queen etc., Proc, Natl, Acad.Sci.USA 86:10029-10033; 1989), EGF-R ELISA (Kettleborough etc., Prot.Engin.4:773-783,1991; Carter etc., Proc.Natl.Acad.Sci.USA.89:4285-4289,1989), carcinomebryonic antigen (Bosslet etc., Br.J.Cancer 65:234-238,1992) and various virus (Tempest etc., Biotechnology 9:266-271,1991; Co etc., Proe.Natl.Acad.Sci.USA 88:2869-2873,1991; Maeda etc., Humanantibodies and hybridomas 2:124-134,1991).
Better generation monoclonal antibody method of the present invention is, transgenic animal are carried out immunity, this animal be manipulated into can The expressed people's antibody.Some examples of this technology are disclosed by (Nature368:856-859,1994) such as Green etc. (Nature Genetics 7:13-21,1994) and Lonberg.By with this transgenic animal of peptide of the present invention immunity, and adopt the hybridoma technology of above-mentioned standard, can produce people's monoclonal antibody.This people's antibody very is applicable to woman's passive immunization.
Pregnancy vaccine of the present invention contains the above-mentioned peptide or the antibody of effective immunogen dosage, and carrier that can be medicinal.Here said " effectively immunogen dosage " is meant and can brings out the dosage of the immunne response with contraceptive effect on female mammal.The dosage of peptide or antibody depends on kind and age, general health situation and the diet of route of administration, Time of Administration, described jenny.
Generally, can use the dosage of per kilogram of body weight 0.01～1000 μ g peptide, the dosage of 0.5～500 μ g peptide/kg body weight is better, and the dosage of 0.1～100 μ g peptide/kg body weight is better.
For a person skilled in the art, carrier that can be medicinal is well-known, for example, comprises disinfection salt solution, lactose, sucrose, calcium phosphate, gelatinum, dextrin, agar, pectin, peanut oil, sweet oil, sesame oil and water.
Vaccine of the present invention can optionally contain one or more adjuvants.Be used as the nonspecific stimulation agent at these adjuvants, to bring out or enhancing immunity is replied.Suitable adjuvant comprises: aluminium hydroxide, aluminum phosphate, white garnet, tocopherol (tocophenols), single phosphenyl lipoid A, Muramyl dipeptide and the saponin such as QuillA.The addition of adjuvant depends on the character of adjuvant self.
In addition, in order to strengthen the immunogenicity of peptide of the present invention, described peptide can be connected with a kind of immunogenic carrier albumen, as tetanus virus (TT) and KLH.Described connection can be undertaken by the reactive group that peptide self exists, or by introducing such reactive group at the C-of this peptide end, as an outer cysteine residues.
This paper said " immunogenic carrier albumen " is meant to have very strong immunogenic protein, and it can trigger host's immunity system, thereby improves the immunogen effect of peptide of the present invention.This immunogenic carrier albumen is different from above-mentioned carrier that can be medicinal on function, therefore, the two is not obscured.
In addition, vaccine of the present invention can also comprise one or more stablizers, and for example carbohydrate comprises sorbyl alcohol, N.F,USP MANNITOL, starch, sucrose, dextrin and glucose; Protein is as albumin or casein; And damping fluid, as alkaline phosphate.
Any female mammal can be accepted the processing of pregnancy vaccine of the present invention.Particularly the woman can accept the processing of vaccine of the present invention.Can therapeutic regimen be optimized according to the inoculation method of standard.The administrated method that is suitable for comprises: intramuscularly, subcutaneous injection, intravenous injection or peritoneal injection, oral or nasal cavity sprinkling.
Description of drawings is as follows:
Fig. 1
Expression monoclonal antibody ZP4A and different peptide bonded ELISA experiments represent with the optical density(OD) form under the 450nm wavelength.The aminoacid sequence of described peptide as SEQ ID NO:2,7 and 9-11 shown in (the corresponding 1-5 that is numbered in Fig. 1).
Fig. 2
The expression monoclonal anti is stopped the inhibition that is subjected to by the peptide QPLWLLQG (SEQ ID NO:2) of round dot representative that combines of ZP4A and the titer plate that scribbles the ZP3 that recombinates, and be not subjected to test by the ELISA of the inhibition of the control peptide of triangle representative, this experiment repeats twice, and what provide is mean value.It is in conjunction with representing with the optical density(OD) form under the 450nm wavelength.
Fig. 3
With monoclonal antibody people's zona pellucida is carried out immunofluorescence dyeing.This immunostaining effect uses the time shutter of being measured by a photomultiplier to represent.Used antibody is: 1=NP11-1A (contrast IgG1); 2=ZP4A.This experiment repeats to do 3 times.Provided mean+SD.
Fig. 4
Suppress combining of people's sperm and human hyaloid band with monoclonal antibody.Antibody test: 1=NP11-1A (contrast IgG1); 2=ZP4A.Provided with 4 average sperm count ± standard deviations of human oocyte bonded.
Fig. 5 represents the result with peptide QPLWLLQG (SEQ ID NO:2) inoculation marmoset. last figure represent through initial immunity (P) and first behind the booster immunization (B) to the antibody titer (representing) of immune peptide with to the antibody titer (representing) of people's reorganization ZP3 with round dot with circle.Figure below is represented the normal ovarian function of a kind of animal of demonstrating of being represented by the plasma progesterone content cycle, consequently by before inoculation and the radioimmunoassay determination that carried out after beginning to inoculate.
Example 1
Produce synthetic peptide by the method (Int.J.Pept.Prot.Res.35:160-214,1990) that Fields and Noble disclose with the solid phase synthesis mode, and utilize glutaraldehyde that the synthetic peptide is connected with bovine serum albumin (BSA).In brief, 100 μ l peptides (1mg/ml) are mixed with 25 μ lBSA solution (12mg/ml), add 25 μ l 60mM glutaraldehyde then.After at room temperature cultivating 1 hour, add 15 μ l 0.5M glycine (pH8.0), after at room temperature placing 30 minutes, by chromatography (PD-10 post, Pharmacia) purifying compound protein fraction.At 0.05M sodium carbonate/bicarbonate damping fluid (coating damping fluid), it is 1 μ g/ml that the peptide that will put together among the pH9.6 further is diluted to concentration, with the consumption coating polystyrene microtiter plates in 100 μ l/ holes, at room temperature spends the night.Two kinds of peptides with aminoacid sequence shown in SEQ ID NO:2 and 7 and 3 kinds of control peptides have been synthesized with this method with aminoacid sequence shown in the SEQ ID NO:9-11.(phosphate buffered saline(PBS)+0.05%Tween 20 at PBST, pH7.5) described peptide is carried out immunodetection with 1 hour time in, adopt during detection be by the ECACC preservation, preserving number is that 94032402 the 1 μ g/ml monoclonal antibody (hereinafter to be referred as mAb ZP4A) that hybridoma cell line produced is carried out.After through 3 washings carried out with PBST of step, the dilution proportion sheep-anti--mouse IgG-horseradish peroxidase conjugate in PBST with 1: 5000 is dropped on the described titer plate, and cultivated 1 hour with the consumption in 100 μ l/ holes.With PBST washing 1 time, wash with water after 2 times, with consumption adding tetramethyl benzidine (TMB) substrate buffer solution in 100 μ l/ holes, and at room temperature cultivated 15-30 minute.Add 100 μ l 4N sulfuric acid (H
2SO
4) the termination staining reaction.The photoabsorption of gaging hole under the 450nm wavelength.Result shown in Figure 1 shows, mAb ZP4A can specificity ground identification peptide of the present invention (numbering of peptide is respectively 1 and 2), and finds combine (numbering of control peptide is respectively 3～5) with control peptide.
Example 2
The Chinese hamster ovary cell that can produce recombinant human ZP3 (Van Duin etc., Biol.Reprod. (waiting to deliver), 1994) supernatant liquor with serum-free applies polystyrene microtiter plates.For this reason, with applying damping fluid (seeing example 1), and at room temperature cultivate this titer plate spend the night (100 μ l/ hole) with the dilution proportion of this substratum with 1: 10.After PBST washing titer plate 3 times, carry out common cultivation with the peptide QPLWLLQG (SEQ IDNO:2) and the control peptide of mAb ZP4A (1 μ g/ml) and different concns.
Immunodetection in conjunction with monoclonal antibody ZP4A is carried out like this: with the consumption in 100 μ l/ holes, in order to sheep-anti--mouse IgG-horseradish peroxidase (HRP) the conjugate titration described titer plate of 1: 5000 dilution proportion in PBST, and cultivated 1 hour.Once with the PBST washing, wash with water after 2 times, with the consumption adding tmb substrate damping fluid in 100 μ l/ holes, and at room temperature cultivated 15-30 minute.Add 100 μ l 4N sulfuric acid (H
2SO
4) to end this staining reaction.Repeat the photoabsorption 2 times of gaging hole with the wavelength of 450nm.
The result as shown in Figure 2, the result shows, peptide of the present invention can suppress mAb 4A and people the combining of ZP3 of recombinating competitively, and this combination is not subjected to the inhibition of control peptide.Can draw such conclusion: on reorganization ZP3 and peptide of the present invention, all exist and to show that by the epi-position of mAb ZP4A identification with peptide of the present invention be the antibody that antigen produces, can go up the reaction of corresponding antigen determinant with ZP3.
Example 3
Obtain human oocyte with external fertilization method.In brief, the ovocyte (confirming that by lacking the spilting of an egg it is unfertilized) with the fertilization failure is kept at ovocyte preservation liquid (OSS, 20mMHEPES, 1.55M MgCl
26H
2O, 0.1% dextran, 10% glycerine, 0.1% polyvinylpyrrolidone, pH7.2-7.4) in, and preserve stand-by down at 4 ℃, be used to people's ovum-fluorometric analysis (hEFA ' s) before, pass through at 3 250 μ lPBS-EFA (PBS, 0.5% bovine serum albumin (BSA), 1% polyvinylpyrrolidone-40,100U/ml penicillin with micropipet, 100 μ l/ml Streptomycin sulphates) the described ovocyte of mode rinsing that shifts in, i.e. zona pellucida.For study mAb ZP4A whether the identification of specificity ground be positioned at ZP3 molecule on the matrix of the ZP1 that has human hyaloid band simultaneously and ZP2, at the volume that contains 100 μ g/ml antibody is the PBS-EFA drop (sample 2 of 25-30 μ l, cultivate ovocyte Fig. 3), under 37 ℃, in adding wet tank, cultivated 1 hour.In order to measure non-specificity bonded level, (sample 1 Fig. 3) is cultivated ovocyte together, and this antibody is the irrelevant antibody of similar isotype with monoclonal antibody NP 11-4-A1 equally under these conditions.Wash 3 times with 250 μ lPBS-EFA with 5-10 minute interval, each washing was all carried out before carrying out following cultivation: be added with the rabbit of puting together with FITC (fluorescein isothiocyanate) anti--cultivate (dilution in 1: 100 among 25～30 μ l PBS-EFA of mouse immuning ball protein, under 37 ℃, in adding wet tank, cultivated 1 hour).Wash again 3 times with the drop of 5-10 minute interval, afterwards ovocyte is introduced in the anti-solution (Johnson, J.Immunol.Meth.43:349-350,1981) that fades of 250 μ l with 250 μ l.For avoiding occurring the phenomenon of fading when in the end analyzing.Each ovocyte is fixed on the slide glass successively, is coated with a ringlet Vaseline on every side, and seal with cover glass.Analyze each ovocyte by fluorescent microscopy.After shining described ovocyte, break forth a time shutter that photo is required with a photomultiplier record with fluorescence at once.In the method, can reflect and this ovocyte bonded antibody amount by the measured time shutter of photomultiplier.The long time shutter is represented not have or antibodies seldom, and can produce the short time shutter with the specific combination of ovocyte.
Result shown in Figure 3 shows, with 5mAb ZP4A cultivator ovocyte, i.e. zona pellucida can produce the fluorescence of higher level, occurs fluorescent dye hardly but cultivate with the contrast monoclonal antibody NP 11-4-1A of isotype.Can draw to draw a conclusion by above result: the epi-position that mAb ZP4A is arranged on natural human ZP3 albumen.
Example 4
Ovocyte according to example 3 described method rinsing people's salt is preserved is adding human body uterine tube liquid culture medium (the HTF-substratum of 0.3% human serum albumin then; See Fertil.Sterll 44:493-498 such as Quinn, 1985) drop in the washing 3 times.The each analysis used 4-5 human oocyte, is that of 25 μ l is contained test compound at volume, and (sample 1: isotype contrasts monoclonal antibody NP 11-4-1A to promptly analyzed antibody; Sample 2:mAbZP4A is 250 μ g/ml) the HTF-substratum in cultivated altogether 1 hour.This cultivation is under 37 ℃ of temperature, has 5%CO
2In adding wet tank, carry out under the condition.The described ovocyte of washing is 3 times in 250 μ l HTF-substratum drops, is collected in then in the volume of 25 μ l, and mixes with 25 μ l HTF substratum, contains 2 * 10 in this substratum
6Individual active people sperm/ml by the Percool purifying, oneself capacitation 3 hours at least under 37 ℃ of described sperm.Cover the cultivation drop of 50 μ l with paraffin oil, and under 37 ℃, 5%CO is being arranged
2Incubator in cultivated 4 hours.
By a dextran gradient centrifugation step separation and combination sperm and free sperm.With the fixing of precipitation+100 μ l supernatant liquors and 1ml, staining fluid [(see: Daniel.J.C.jr by 0.9ml BWW-substratum, (work), Methods of Mammalian, Embryology, W.H.Freemman, San Fransisco, PP.86-116,1971), 0.1ml 10% glutaraldehyde, 1-10 μ l 2mg/ml bismenzimid H33258 (Hoechst)] mix, and 4 ℃ of following overnight incubation.At last, from this solution, collect ovocyte, in the anti-solution that fades, cultivate, and be fixed on the slide glass by example 3 described methods with microscope and little apparent transfer pipet.Measure in conjunction with sperm number by fluorescence microscopy, and count every mm
2The fixing average cell number on two of the upper and lower surface of ovocyte focusing surfaces.
This result of experiment as shown in Figure 4.Opposite with the contrast monoclonal antibody, mAb ZP4A can suppress combining of people's sperm and human oocyte.Therefore, by with peptide of the present invention being the antibody capable and the natural zona reaction on the human oocyte that antigen produces, and the combining of inhibition sperm and zona pellucida, thereby bring out sterile.
Example 5
With peptide QPLWLLQG (SEQ ID NO:2) female marmoset (Callithrixjacchus) is carried out immunity.At first on the C-of this peptide end site, connect a cysteine residues, for this peptide provides a reaction site.Is that Toxoid,tetanus (TT) is connected with sulfo group-SMCC (N-succinimido 4-(N-maleimide methyl) hexanaphthene-1-carboxylic acid) with this reaction site and the carrier proteins of described peptide.At first, the 4mg peptide is dissolved in 5ml0.1M phosphoric acid buffer pH6.0, among the 5mM EDTA,, can reaches-the SH base so that on this peptide, form to wherein adding 6mg 2 mercapto ethanol amine.Cultivated 1 hour down at 37 ℃, then by this peptide of Sephadex G-25 chromatography purification, and 4 ℃ of preservations down.
The activation of carrier proteins is carried out like this: this albumen of 4mg is dissolved in the 5ml 50mM sodium borate buffer liquid, and pH7.0 adds 2mg sulfo group-SMCC then, and cultivates 1 hour down at 30 ℃.For this albumen of purifying, adopt Sephadex G25 chromatography.To contain the proteic fraction of activated carrier and be collected in together, and be concentrated into 5ml with an Amicon B-15 microconcentrator.In order to put together, the described fraction that contains following peptide to be added activatory concentrate among the TT, and under 4 ℃, in vibrator, cultivated 20 hours.Subsequently with puting together material more than the Sephacryl S-400 column purification, this post 0.1M Tris HCl, 1mM MgCl
2PH7.0 balance mistake, flow velocity are 20ml/h.
Immunity: before immunity 3 months, the menstrual cycle of monitoring marmoset, and survey a serum progesterone content by per two weeks and confirm its normal ovarian function.During initial immunity, the 200 μ g conjugates that will be dissolved among the PBS by subcutaneous injection divide the injection of several places, and cumulative volume is 0.2ml.Used PBS non-ulcerative Freund ' the s adjuvant emulsion mistake that contains 250 μ g N-acetyl muramyl-L-alanyl-D-isoglutamines (MDP).Prepare booster shots liquid with similar approach, different is to have removed MDP.
For the immunne response and the ovarian function of monitoring immune animal, draw blood weekly 2 times, measure anti--peptide QPLWLLQG antibody and anti-reorganization ZP3 antibody respectively, and serum progesterone content.
Reply with measuring anti--peptide through maleic anhydride (maleic anhydrade) activatory titer plate (Pierce).This titer plate applies (100 μ l/ hole), and spends the night under room temperature (RT) with 5mg peptide/ml PBS pH8.0.Pour out after the described peptide solution, seal damping fluid (3%BSA, 0.05%Tween 20, are dissolved among the PBS) washing hole 2 times, under RT, cultivated 1 hour with other 200 μ l sealing damping fluid with 200 μ l.Through after above-mentioned the 3rd washing step, 2 times of diluents of testing sample to be added, dilution range is 1/10～1/20480 (75 μ l, 2 hours, room temperature).With lavation buffer solution (0.1%BSA, 0.05%Tween 20, are dissolved among the PBS) washing 3 times, then 100 μ l are resisted-monkey HRP conjugate (dilution 1/1000) to be added in each hole, under RT, cultivated 1 hour.To the further processing of titer plate as described in the example 1.
Roughly discern the recombinate ELISAs of ZP3 of people by example 1 described method.
Measure marmoset plasma progesterone content with non-extraction radio immunoassay.The each analysis done 3 repetitions, uses by the 0.1M phosphoric acid citrate buffer solution (PCB) that contains 0.1% gelatin, and pH6.0 is diluted to the 2.5 μ l blood plasma that cumulative volume is 150 μ l.Add goat-anti-progesterone antibody diluent 100 μ l and 100 μ l iodate progesterone tracer agents of 1/10000 in PCB, and under 25 ℃ with this water culture 3 hours.Add second kind of antibody then, 100 μ l donkeys resist-sheep blood serum (ScottishAntibody Production Unit (SAPU), Scotland) 1/64 times of diluent in PCB, 1/3200 diluent that adds 100 μ l normal sheep serums (SAPU) again, with gained solution 4 ℃ of following overnight incubation.At last, the 0.2%Triton X-100 that add 1ml 4% polyoxyethylene glycol, in 0.9% salts solution, is made into, and at 4 ℃ of centrifugal 30 minutes of 1500 * g down.Go up counting at γ more than (multigamma) counter (LKB) after outwelling supernatant liquor.
Result shown in Figure 5 shows, can bring out immunne response with peptide immunity of the present invention.Complete people ZP3 albumen and the peptide QPLWLLQG (last figure) of antibody capable identification that is produced, and the plasma progesterone content cycle shows that it has normal ovarian function.Sequence table: (1) general information:
(i) applicant:
(A) name: AKZO NOBEL N.V.
(B) street: Velperweg 76
(C) city: Arnhem
(E) country: Holland
(F) postcode: 6824 BM
(G) phone: 04120-66376
(H) fax: 04120-50592
(I) fax: 37503 akpha nl
(ii) denomination of invention: new contraception peptide
(iii) sequence number: 11
(iv) computer-reader form:
(A) media type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, the information of Version#1.25 (EPO) (2) SEQ ID NO:1:
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:1:
Pro?Leu?Trp?Leu?Leu?Gln
The information of 15 (2) SEQ ID NO:2:
(i) sequence signature:
(A) length: 8 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:2:
Gln?Pro?Leu?Trp?Leu?Leu?Gln?Gly
The information of 15 (2) SEQ ID NO:3:
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:3:
Pro?Leu?Trp?Phe?Trp?Gln
The information of 15 (2) SEQ ID NO:4:
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:4:
Pro?Met?Trp?Thr?Leu?Gln
The information of 15 (2) SEQ ID NO:5:
(i) sequence signature:
(A) length: 8 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:5:
Pro?Gln?pro?Leu?Trp?Leu?Leu?Gln
The information of 15 (2) SEQ ID NO:6:
(i) sequence signature:
(A) length: 8 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:6:
Pro?Leu?Trp?Leu?Leu?Gln?Gly?Gly
The information of 15 (2) SEQ ID NO:7:
(i) sequence signature:
(A) length: 20 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:7:
Leu?Cys?Tyr?Pro?Gln?ProLeu?Trp?Leu?Leu?Gln?Gly?Gly
1 5 10
Ala?Ser?His?Pro?Glu?Thr?Ser
The information of 15 20 (2) SEQ ID NO:8:
(i) sequence signature:
(A) length: 15 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:8:
Ala?Asp?Gly?Ala?Pro?Met?Trp?Thr?Leu?Gln?Gly?Ala?Ala
1 5 10
Gly?Ala
The information of 15 (2) SEQ ID NO:9:
(i) sequence signature:
(A) length: 20 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:9:
Lys?Val?Thr?Leu?Ala?Glu?Gln?Asp?Pro?Asn?Glu?Leu?Asn
1 5 10
Lys?Ala?Cys?Ser?Phe?Ser?Lys
The information of 15 20 (2) SEQ ID NO:10:
(i) sequence signature:
(A) length: 21 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:10:
Thr?Asp?Asp?Ala?Leu?Val?Tyr?Ser?Thr?Phe?Leu?Leu?His
1 5 10
Asp?Pro?Arg?Pro?Val?Gly?Asn?Leu
The information of 15 20 (2) SEQ ID NO:11:
(i) sequence signature:
(A) length: 18 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: peptide
(xi) sequence statement: SEQ ID NO:11:
Pro?Ser?Asp?Thr?Ser?Val?Val?Leu?Leu?Gly?Val?Gly?Leu
1 5 10
Ala?Val?Val?Val?Ser
15
Claims (19)
1. the peptide that has the aminoacid sequence of forming by 8-50 amino-acid residue, this peptide comprises aminoacid sequence PLWLLQ (SEQ ID NO:1) or its analogue at least, prerequisite be described analogue can with by the ECACC preservation, preserving number is 94032402 the monoclonal antibody generation immune response that hybridoma cell line produced.
2. peptide as claimed in claim 1 is characterized in that described peptide comprises aminoacid sequence QPLWLLQG (SEQ ID NO:2) or its analogue at least.
3. as the peptide of claim 1 or 2, the aminoacid sequence that it is characterized in that described peptide is QPLWLLQG (SEQ ID NO:2).
4. as the peptide of claim 1 or 2, the aminoacid sequence that it is characterized in that described peptide is PQPLWLLQ (SEQ ID NO:5), PLWLLQGG (SEQ ID NO:6) or LCYPQPLWLLQGGASHPETS (SEQ ID NO:7).
5. as the peptide of claim 1 or 2, the aminoacid sequence that it is characterized in that described peptide is ADGAPMWTLQGAAGA (SEQ ID NO:8).
6. the purposes that each peptide in the claim 1～5 is used to produce antibody.
7. the antibody that produces at each described peptide in the claim 1～5.
8. antibody as claimed in claim 7 is characterized in that described antibody has specificity in conjunction with the ability that comprises the epi-position of aminoacid sequence PLWLLQ or QPLWLLQG.
9. as the antibody of claim 7 or 8, it is characterized in that described antibody is monoclonal antibody.
10. antibody as claimed in claim 9 is characterized in that described antibody is that 94032402 hybridoma cell line is produced by ECACC preservation, preserving number.
11. produce clone as the antibody of claim 7～10.
12. by the ECACC preservation, preserving number is 94032402 hybridoma cell line.
13. will as in the claim 1～5 each peptide and/or as each antibody in the claim 7～10 as the purposes of therapeutic substance.
14. medicinal compositions contains one or more as each peptide or one or more antibody as claim 7～10 of claim 1～5, and a kind of carrier that can be medicinal.
15. pregnancy vaccine, contain one or more as in the claim 1～7 each peptide or one or more as each antibody in the claim 7～10, and a kind of carrier that can be medicinal.
16. be used for detecting the method for patient's serum autoimmune antibody, may further comprise the steps:
A) in patient's body, obtain serum sample;
B) allow described sample contact as each peptide in the claim 1～5 with one or more, and selectively with a kind of diagnostic reagent contact that contains one or more antibody of the present invention,
C) determine the existence of described autoimmune antibody by suitable detection reaction.
17. will as in the claim 1～5 each peptide or as each antibody in the claim 7～10 as the purposes of diagnostic substances.
18. test kit that is used for the autoimmune antibody of test samples, described test kit contains one or more as each peptide in the claim 1～7, also contain a kind of diagnostic reagent selectively, this diagnostic reagent contains the monoclonal antibody just like claim 7～10.
19. a diagnostic reagent, it contain that useful a kind of detection agent mark crosses as each monoclonal antibody in the claim 7～10.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94202392 | 1994-08-22 | ||
| EP94202392.0 | 1994-08-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1158623A true CN1158623A (en) | 1997-09-03 |
Family
ID=8217113
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95195212.9A Pending CN1158623A (en) | 1994-08-22 | 1995-08-18 | New immunocotraceptive peptides |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0777688A1 (en) |
| JP (1) | JPH10505340A (en) |
| KR (1) | KR970705578A (en) |
| CN (1) | CN1158623A (en) |
| AU (1) | AU3471395A (en) |
| BR (1) | BR9508748A (en) |
| CA (1) | CA2198265A1 (en) |
| FI (1) | FI970744A (en) |
| HU (1) | HUT77262A (en) |
| WO (1) | WO1996006113A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19622289A1 (en) * | 1996-05-23 | 1997-11-27 | Schering Ag | Zona Pellucida Proteins for contraception |
| EP0923602A4 (en) * | 1996-07-03 | 2005-06-15 | Zygam Inc | Immunocontraceptive compositions containing sperm antigens, and methods of use |
| US7037663B2 (en) | 1998-02-19 | 2006-05-02 | Eastern Virginia Medical School | Human zona pellucida protein 3 and uses thereof |
| DE69922489T2 (en) | 1998-02-19 | 2005-12-15 | Eastern Virginia Medical School | Recombinant active human zona pellucida |
| WO2003011118A2 (en) | 2001-08-02 | 2003-02-13 | Trinity Biomedical Technology Corporation | Human zona pellucida proteins and methods of their use in diagnosing male infertility |
| CN101906163B (en) * | 2009-06-05 | 2012-06-27 | 上海交通大学医学院 | Immunocontraceptive synthetic peptide and immunocontraceptive chimeric peptide and application thereof |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4996297A (en) * | 1987-10-07 | 1991-02-26 | Zonagen, Inc. | Recombinantly expressed rabbit zona pellucida polypeptides |
| CA2058999A1 (en) * | 1989-06-12 | 1990-12-13 | Jurrien Dean | Contraceptive vaccine based on cloned zona pellucida gene |
| HUT64394A (en) * | 1990-08-27 | 1993-12-28 | Akzo Nv | Protein of human zona pellucida zp3 |
| JPH05176771A (en) * | 1991-11-29 | 1993-07-20 | Tonen Corp | Dna cording pig egg clear zone pzp-3 |
| JPH0614784A (en) * | 1991-11-29 | 1994-01-25 | Tonen Corp | Dna coding feline ovum clear zone fzp-3, its expressing peptide, and contraceptive vaccine containing the pfptide |
| JPH05336974A (en) * | 1991-12-26 | 1993-12-21 | Tonen Corp | Dna capable of coding canine egg zona pellucida czp-3, czp-3-related peptide coded by the same dna and contraceptive vaccine containing the same peptide |
| JPH06179698A (en) * | 1992-12-15 | 1994-06-28 | Tonen Corp | Peptide related to clear zone pzp-3 of pig egg |
| JPH06217777A (en) * | 1993-01-29 | 1994-08-09 | Tonen Corp | Dna sequence-coding cat egg transparent zone fzp2 |
-
1995
- 1995-08-18 KR KR1019970701103A patent/KR970705578A/en not_active Application Discontinuation
- 1995-08-18 AU AU34713/95A patent/AU3471395A/en not_active Abandoned
- 1995-08-18 BR BR9508748A patent/BR9508748A/en not_active Application Discontinuation
- 1995-08-18 JP JP8507795A patent/JPH10505340A/en active Pending
- 1995-08-18 CN CN95195212.9A patent/CN1158623A/en active Pending
- 1995-08-18 CA CA002198265A patent/CA2198265A1/en not_active Abandoned
- 1995-08-18 WO PCT/EP1995/003311 patent/WO1996006113A1/en not_active Application Discontinuation
- 1995-08-18 HU HU9701726A patent/HUT77262A/en unknown
- 1995-08-18 EP EP95931169A patent/EP0777688A1/en not_active Ceased
-
1997
- 1997-02-21 FI FI970744A patent/FI970744A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| KR970705578A (en) | 1997-10-09 |
| FI970744A0 (en) | 1997-02-21 |
| HUT77262A (en) | 1998-03-02 |
| EP0777688A1 (en) | 1997-06-11 |
| JPH10505340A (en) | 1998-05-26 |
| CA2198265A1 (en) | 1996-02-29 |
| WO1996006113A1 (en) | 1996-02-29 |
| MX9701304A (en) | 1998-05-31 |
| AU3471395A (en) | 1996-03-14 |
| FI970744A (en) | 1997-02-21 |
| BR9508748A (en) | 1997-08-12 |
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