JPH06500690A - Human egg zona pellucida protein ZP3 - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Human protein ZP3 The present invention relates to a polypeptide that has human ZP3 activity or at least partially human ZP3 antigenicity. It relates to a lipeptidic acid or a functional derivative thereof.

During the fertilization process, the first interaction between mammalian gametes occurs when the egg surrounds the female gamete. Mediated by binding of sperm cells to species-specific receptors on the zona pellucida (ZP) . ZP is an extracellular matrix containing three glycoproteins ZPI, ZP2 and ZP3. of which ZP3 has been identified as a sperm receptor (Wassarm an, Development 108. 1-17; 1990).

Numerous in vitro and i The n vivo research report shows that ZP3 could be used in experimental strategies aimed at developing immunocontraceptives. It has been shown that it is an important target antigen candidate for cancer (Henders).

n et al, J, Reprod, Fert, 83°325-343; 1 988 and references thereof), and recently a mouse ovary cDNA expression library was Mouse ZP3 cDNA was detected by screening with mouse ZP3 antibody. rowing and characterization, representing an important advance toward this goal (Ringu ette et at.

Developmental Biology 127゜287-295; 1 988), then ZP3c to monolayer the corresponding genomic ZP3 DNA. DNA probes were used (Chamberlin and Dean, De velopmental Biogy 131, 207-214. 198 8; K1n1ochet al, Proc, Natl, Acad, Sci, USA 85.　6409-6413. 1989).

The contraceptive ability of ZP3 was demonstrated by the use of an oligopeptide derived from the mouse ZP3 amino acid sequence. Backed by studies demonstrating long-term infertility in female mice after vaccination (Mtllar et al, 5science 246°935-93 8. 1989).

Because the ZP3 sperm receptor is species-specific, mouse polypeptides may be used for immune evasion in humans. Not suitable for pregnancy. Furthermore, polypeptides of non-human origin may pose unwanted immune responses to the human body. may cause an epidemic response. Therefore, a safe and effective method based on (part of) the human sperm receptor. To develop a contraceptive vaccine, information about the human ZP3 polypeptide and its It is necessary that the polypeptide be available.

It is naturally impossible to isolate sufficient amounts of human ZP3 polypeptide from female gametes. It is. However, we have identified a human gene and elucidated its sequence. It was very successful. Thus, by recombinant DNA technology or facial polypeptide synthesis, Ability to produce ZP3 polypeptides and/or ZP3 polypeptide fragments Sexuality has expanded.

Therefore, the present invention provides novel D It concerns NA molecules. The DNA molecule contains at least a portion of the sequence shown in FIG.

The present invention further provides polypeptides encoded by at least a portion of the above DNA molecules. This also applies to de. The polypeptide comprises at least a portion of the amino acid sequence shown in FIG. .

It will be appreciated that functional derivatives and fragments of the polypeptides of the invention are also included. A functional derivative included in the invention is one in which one or more of the amino acids is chemically equivalent. refers to a polypeptide substituted with a amino acid.

The most important point is, of course, that it exhibits human ZP3 antigenicity.

This is an antibody that recognizes human ZP3 after administration (optionally with an adjuvant). It means to produce.

The polypeptide of the present invention can be produced synthetically or by recombinant DNA technology. Methods for producing synthetic polypeptides are well known to those skilled in the art. Therefore, there is no need to elaborate.

The production of polypeptides by recombinant DNA technology involves a number of 0 The polypeptide to be expressed is a DNA sequence, or more precisely encoded by a nucleic acid sequence.

This nucleic acid sequence must be (optionally) transcribed and translated into the desired polypeptide. To reach this goal, the nucleic acid sequence is usually cloned into a vector. to transform host cells. The vector may be self-replicating, or the vector may be of the host's DNA. It may be incorporated into A.

Different host cells result in different polypeptides.

Prokaryotic cells do not have the organelles necessary for glycosylation.

Polypeptides produced by prokaryotic cells do not have carbohydrate side chains; eukaryotic cells have Yeast cells have a glycosylation mechanism that differs from mammalian cells. Give a turn.

The expression system that gives the most "natural" glucosylation pattern is the polypeptide of the present invention. suitable for Mammalian cells are optimal for this purpose.

The inventors also identified an epitope on the human ZP3 polypeptide that may have contraceptive potential. Ta. These epitopes span at least a portion of the following sequences:

ln- to 5erArgArgGlnProHisValMatSar.

-GluVaIG1yLauHisGlucy! iGlyAsnsarM@tG lnValThrAspAspAlaLeu-.

-PhaSerLauArgLauMetclucluAsn’rrpAsnA laGlul, ysArgsel: ProThrPhe-.

-CysGlyThrProsarHisserArgArgGlnProHi sValMatserGlnTrpsar-.

7 bows Small polypeptides such as these epitopes are easily produced synthetically . Such polypeptides bind to the same or different "epitopes" to generate larger A polypeptide with higher antigenicity may be formed.

It is an object of all the polypeptides of the invention to use these polypeptides to create contraceptive vaccines. The goal is to manufacture products. Vaccination may be passive or active immunization.

In the case of active immunization, the polypeptide of the invention (optionally with an adjuvant) ) administered to women. As a result of administration, the woman develops an immune response. Human ZP on egg cells Antibodies that recognize 3 are produced. These antibodies prevent the sperm from binding to the sperm. Either the antibody binds specifically to the receptor binding site, or the antibody blocks this binding by steric hindrance. Stop. Passive immunization is basically performed in the same way. instead of antigen or its analogue , directly administering antibodies against the antigen. Therefore, antibodies directed against the polypeptides of the invention The antibody must be

This is accomplished by appropriate mammalian active immunization procedures.

Mammalian B lymphocytes are harvested after an appropriate time and immortalized by fusion or transformation. let These methods are well known to those skilled in the art. From immortalized lymphocyte cultures Antibodies can be isolated.

However, antibodies of animal origin have problems. Repeated administration may result in immunization Women may develop an anti-antibody response. Therefore, the human body does not produce an immune response. It is preferred to use adapted antibodies or small portions of antibodies. Antibiotics like CDR transplantation Methods of adapting the body to the human body are known (J.

nes et al., Nature 321, 522-525. 1986 ), methods for producing antibody fragments specific for the antigen of the original antibody are also known ( Udaka et al., Molec, Immunol, 27.25-35.　 (1990), which did not generate an antigenic response to antibodies against the polypeptide of the present invention. For this purpose, human antibodies or fragments or derivatives thereof can also be used. .

Human antibodies can also be produced by in vitro stimulation of isolated B lymphocytes. and obtained from a woman immunized with at least one polypeptide of the invention. (Immortalized) B lymphocytes can also be isolated from B lymphocytes. Another object of the present invention is to Use of ZP3 voliberatide and antibodies against the polypeptide in test kits It is for use.

Currently, several types of antibodies against human ZP3 have been obtained.

Anti-idiotypes that recognize the antigen-binding site of an antibody and are therefore an "internal image" of the antigen It is within the knowledge of those skilled in the art to produce antibodies.

These anti-idiotypic antibodies are also very useful for vaccine and diagnostic purposes.

In the experimental section below, we will demonstrate the human ZP3 DNA sequence and human ZP3 amino acid sequence. and methods for expressing recombinant polypeptides exhibiting ZP3 antigenicity, as well as methods for expressing such antigens. These examples are intended to be illustrative only; However, it is not intended to limit the scope of the invention.

and 11 Company 1juL Tate Niichi All recombinant DNA procedures were performed essentially according to procedures known per se (Samb rook et al, M.

1ecular Cloning, a 1laboratory manual, 2nd edition, C3H-1 laboratory press, 1989>.

Restriction enzymes and DNA modifying enzymes were used as recommended by the manufacturer.

Human ovarian gtlo cDNA library was purchased from Stratagene. .

1 Gonu Shi Tom The oligonucleotide is Applied Biosystems 381A DN. A synthesizer and used directly for cloning purposes.

Each 1-9” “Precepts” Fields and Noble (rnt, J, Pept.

Prot, Res, 35. 160-214; 1990). Oligopeptides were produced by solid-phase peptide synthesis using a procedure described below. Z below P3 peptide was synthesized (amino acids are indicated by three letter code).

1 good 1 [ Polyclonal antisera were generated using various antigens.

-hha, human egg zona pellucida preserved in salt was heat-solubilized and used for rabbit immunization. Mixed with Freund's adjuvant. EL coated with pig ZP protein Antiserum titers were assayed with ISA alates.

-Ga1-ZP3 protein, the fusion protein is sonicated based on its insolubility. It was partially purified from treated bacteria and subjected to SDS polyacrylamide gel electrophoresis (SD S-PAGE). Nitrogenizing proteins by electroplotting Transferred to cellulose membrane. Hybrid Tanno (cut out the region containing lithium and DM Dissolved in SO. Mix this with Freund's adjuvant in a ratio of 1:1, and It was used for immunization of guinea pigs.

-CH0ZP3. Produced by Chinese Hamster Ovary (CHO) cells A similar procedure was performed for ZP3 protein. The concentrated culture medium was transferred to 5DS-PAG. Separated by E. A region of 40 to 60 kilodaltons was excised and used for immunization of mice. used.

Ichiomishi full (z Kamishikai-0) The synthesized peptide is keyhole rimpe 7 tohemoshi physically conjugated to anin (KLH) and injected into rabbits. K in rabbits as a control immunized with LH alone. ELISA microtiter with serum coated with peptide Screened on plates.

Human assay.

Unfertilized human eggs preserved in salt (48 hours with sperm in IVF program) After incubation), dilute the antiserum with wig solution A [PBS + 5% BSA]. : diluted to 50). After 3 washes, the eggs are washed with a second anti- body (pig anti-rabbit or mouse conjugated to FITC and diluted in 7:1:100 of M street solution A). (mouse antibody) for 1 hour at 37°C. After washing three more times, Ni Fluorescence was measured using a radiometer and an irradiation analyzer. Unstained (negative) Control eggs remained dark even after long irradiation, while positively stained egg zona pellucida responded to short irradiation times. .

Human-BΔ assay Human eggs (described above) were incubated in rabbit serum and buffered (BWW + 3% B Wash three times with SA) and incubate in buffer (control) or antibody (undiluted, 37°C, 1 hour). Incubated, washed 3 times, and fertilized human sperm (107 cells/ml, 3 7°C for 16 hours) was added dropwise. 5 freely by repeated pipetting Adhering sperm were removed from the eggs. Fix the bound sperm and add 1% glutaralde. Dyeing with BWW containing Hyde and Hoechst (H33258, 20g/ml) The number of bound spermatozoa was counted using a fluorescent microscope.

1" Mouse ZP3 probe mechanism By linking 8 synthetic oligonucleotides (27-5omer), 13 A probe was constructed with the 5bP mouse ZP3 probe. Exons 5 and 6 of the mouse gene (Ringuette et al.).

Developmental Biology 127゜287-295; 1 988) with a single 5' BamHI and 3' Hin dl II restriction site and subcloned into pGEM3 (Promega). I did a ning. The nucleotide sequence was then confirmed.

Cloning of human ZP3 gene and human genome EMBL3 library Screening was carried out using the P-Rinshi ZP3 probe (the above 135 bp fragment).

Three overlapping EMBL clones showed strong hybridization. Therefore, we characterized it in more detail. Figure 1 shows the physical restriction map of these clones. The restriction fragments from clone 11 and Dl shown in 1 were inserted into pGEM vector and The human gene was subcloned into used for genome characterization. In this characterization, “P” from the mouse ZP3 sequence Exon positions were determined by Southern blot analysis using I[identical oligonucleotides]. After determining, the sequence was analyzed. As shown in Figure 1, human genes are approximately It consists of eight exons spread over 20kb. All identified exons are bilateral Consensus splice donor and splice acceptor signals (as shown) ). Complete coding of ZP3 gene through sequence analysis of all exons The sequence of the human ZP3 gene was predicted to be 41,437 daltons (Figure 2). encodes a polypeptide consisting of 372 amino acids with a calculated molecular weight of .

Cloning of human ZP3 cDNA Human ovary cD with 32p labeled fragments from human ZP3 exons 1.5 and 7. The NA library (gtlo) was converted into a slightly scratched partial cDNA containing only the side half ( Exons 5-8) were obtained.

Sequence analysis of three independent cDNA clones revealed that one residue in exosif Other than that, it was confirmed that the genome ZP3 sequence was identical to the previously determined genome ZP3 sequence (Figure 2) , G and C residues were detected in the genome and cDNA, respectively, at nucleotide position 21064. It was done. Therefore, in the encoded amino acid sequence, arginine or A threonine residue will be detected; this sequence difference is probably related to the human ZP3 gene. and shows versatility in polypeptides.

CHO Ko4'ZP3's Z for insertion into mammalian expression vectors for expression in CHO cells. Reduce the size of the P3 gene.

Various ZP3 DNA fragments are combined to create a “minigene” as shown in Figure 3. was formed. Exons 1 and 2 were analyzed using PCR technology (Horton et al., G. ene 77, 61-68, 1989; Yon and Fr1ed, Nu cl, Ac1ds Res, 17,4895.1989>, respectively. A 5' EcoRI and 3' XbaI site was created. Then this DNA flag Genome XbaI-9al containing exon 3.4 and part of exon 5 The partial cDNA containing exons 5 to 8 was ligated to the Sal l- ligated to the HlndIr fragment. The 2,7kb r mini-remains formed ZP3 gene contains a truncated intron between exons 2 and 3, and ZP3 exon 3 and The nucleus of this ZP3 construct contains natural introns between exons 4 and 5. The integrity of the polypeptide encoding the leotide information was fully confirmed by sequence analysis. It was.

ZP3 r minigene” was then inserted into the r@mammalian expression vector, and the ZP3 gene Transcription was initiated by the strong SV40 early promoter. Furthermore, this vector , stable transformants can be isolated by selecting for G418 resistance. A selectable marker gene such as aminoglycodylphosphotransferase (C olbere-Garapin et al., J. Mo1. Biol, 150 ．． 1-14. (1981), β for proper RNA processing of expressed genes. - Contains Globin splicing and S40 polyadenylation signals Ta. Calcium phosphate precipitation method (Graham and van der Eb, Virology 52, 456-467.1973; Wiggler et at, , Proc, Natl.

Acad, Sci USA 76, 1373-1376゜1979) CHO cells were transfected with the ZP3 expression construct. G418 resistance transformation Populate a population of cells (representing 300-500 independent clones) with the recombinant ZP3 gene. The expression of offspring was analyzed.

As shown in Figure 4, total RNA from pooled transformed cells was combined with 32p-labeled ZP3. Northern blot analysis using the probe revealed that the highly expressed actin gene (not shown) The expression level of the transfected ZP3 gene (in comparison) was found to be relatively high. found. Furthermore, minigene RNA is a natural transcript present in human ovarian RNA. It has been demonstrated that the mRNA is properly processed into a slightly larger mRNA than the normal one (r f! i49), this size difference is due to the flanking present in the expression vector. This appears to be mainly due to the 5' and 3' sequences.

Furthermore, a partial cDNA containing exons 1 to 5 from G418-resistant CHO transformants Proper splicing and processing of recombinant genes can be achieved by isolating A. was confirmed. The sequence data of this cDNA is similar to the transfected DNA. similar, and the intron is properly spliced from the ZP3 minigene transcript. This supports the conclusion that

The presence or absence of recombinant ZP3 polypeptide in the culture medium of a growing clonal population Tested by Western blot analysis. Bot on deglycosylated porcine ZP3 Reclona ete Re5search 18.　251-265゜1987　 could be used to detect recombinant ZP3 polypeptide in the culture medium. This Gnar was absent in the medium from non-transfected CHO cells.

Nii! 'ZP3's C) (isolated by PCR from O transformed cells for expression in E. coli) cDNA fragment covering exons 1 to 5 and human ovary cDHA library. Using a partial cDNA clone (containing exons 5 to 8) isolated from the library, First, full-length ZP3 cDNA was constructed using the following methods. 4 ZP3 cDNA fragments cloned into the E. coli LacZ gene in pEX plasmid (Biores). Tanley and Luzio, EMBOJ, 3. 1429-143 4. β-galactosidase-ZPB! as reported by 1984 Combine A polypeptide was produced. Expression of the fusion polypeptide in E. coli was determined by SDS port. Reacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis Tested by analysis. The results of these experiments are shown in Figure 5. After heat shock induction, four All of the LacZ-ZP3 elliptic constructs produced fusion polypeptides, but to a lesser extent. It was significantly lower than the LacZ gene. The molecular weight of the hybrid polypeptide is As observed on a Coomassie-stained gel (Fig. 5A), the expressed ZP3 moiety It was a match. This indicates that anti-β-galactosidase antibody (not shown) and KLH Western culture using rabbit antiserum against ZP3 ami7M341-360 bound to This was further confirmed by tan plot analysis (Figure 5B). fused polype containing this region Only peptides (ie ZP3-A and ZP3-D) were recognized by the antiserum. KL Antiserum against H produced results similar to the background staining in Figure 5B (as shown). In addition, the βGa]-ZP3 fusion polypeptide has an effect on all human zona pellucida. recognized by antiserum (not shown).

zp human Δ Human egg fluorescence assay to characterize the binding of various ZP antibodies to the human egg zona pellucida. I went to The results of three different experiments are shown in Figure 6. Although normal mouse serum could not be fluorescently stained, total antiserum against ZP acid component Strong to medium level fluorescence was observed.

As is clear from the illustrated data, ZP3 (93-110).

ZP3 (172-190), ZP3 (327-344).

Antisera against ZP3 (341-360) and ZP3 (362-372) were Recognize oocytes.

1 Child 2 N Elephant A Ball” 1 Zuko Kyoumi Ward 111 L1 ZP3 Antibody Contraceptive Potency Hi I/Sperm-Egg Analyzed by zona pellucida binding assay. The results of this assay are shown in FIG.

Antibodies against whole human egg zona pellucida showed a 75% inhibition rate of sperm-egg zona pellucida binding. K Antiserum against LH does not interfere with sperm-egg binding, whereas ZP3-peptide antibodies (Peptides: ZP3 (93-110), ZP3 (172-190).

The mixture of ZP3 (341-360) and ZP3 (362-372) is 35 % relative inhibition of sperm binding. Antibody against recombinant ZP3 from CHO cells The sperm binding was low at 55%.

! jpi LN15” Hi1 Figure 1 EMBL O-7I 1, D1211"El (A) and h) ZPSi Denji <7) Yes. Restriction site + S, Sal I; B, Bam1ll.

Figure 2 At position 1064 showing the nucleotide and amino acid sequence of human ZP3, G or The C residues found in the genome and cDNA, respectively, are indicated in circles.

Polyadenylation signals are underlined.

Figure 3 2. Demonstrates size reduction of the lP3 gene for insertion into mammalian expression vectors; 7kb ZP3r minigene 1 into the following three types of fragments: A. Frame is extracted by PCR method to create EcoRI-XbaI fragment. exon 1 and exon 2 combined with each other in the system; exon 3.4 and exon 2; Genomic XbaI-SalI fragment with part of protein 5: C, 5alr-HindlII ZP3 cDN^DN with exons 5 to 8 A-ment (with artificial 3' BamHI and HindlII sites) Assembled. The three fragments were isolated from EcoRI and [l1ndlII 2.7kb EcoRiBamH behind the SV40 promoter in the current vector It was placed as an I fragment.

Figure 4 Tolan detection using ZP3 DNA and poly(A)” RNA (5g) derived from human ovary Indicates the appearance.

Lane 3. human ovary.

The entire ZP3 cDNA in pGEM3 was used as a probe.

presentation E, eoli carrying QEX plasmid carrying human ZP3 cDN^DNA Production of β(a1-ZP311 polypeptide in strain POP2136) A cut analysis is shown. From left to right, fill the gel with the following samples: 1 Before induction; + , induction 2-question control pEX: pEX-ZP3^ (KpnI-BamHI flag ment; all cDN^).

pEX-ZP3B (Konl-Sall fragment 1-2 amino acids 1-241) cDNA cut at exon 5 encoding); pEX-ZP3C (Kpnl- Exon 1 coating Sph17 lagmen't-near mi/acid 1-102 cDNA cut at the end).

pEX-ZP3D (Saml-BaIl old fragment, amino acids 257-37 2f! : 3' half of exon 5 encoding exons 6, 7 and 8) molecular weight (M ) marker is on the far left (expressed in kilodaltons). Bacteria are , OD. at 30°C. ,. = 0.5-1, then grown at 42°C for 2 hours. I was induced for a while. Aliquots were removed, centrifuged, and whole cell lysates were analyzed.

Figure 6 Represents the binding of various antibodies to human eggs (measured with materials and Expressed as a percentage (100g = dark.

Oz = fluorescent egg).

3: Anti-serum against KLH; 4 - 8: ZP3 (93-110>, ZP3 (172-190>, Z P3 (327-344>'; ZP3 (341-360) and ZP3 (362- 372); 9: anti-serum against βGa1-ZP3; 10, group A mixture of sera from three mice immunized with modified CHO-producing ZP3; 11: mixture of sera from 3 control mice.

irradiation Light snow time is shown as a percentage of control and standard deviation of the mean.

Figure 7 Inhibition of sperm-egg binding by anti-ZP3 antibody (see Materials and Methods 4) .

1: control; 2: Anti-serum against KLFI; 4: Zr2 (93-110), Zr2 (172-190), Zr2 (341 -360) and human ZP3 (36:-372); 5: Recombinant A mixture of sera from three mice immunized with body CHO-producing ZP3.

vinegar.

material Kotobuki September-E” Fig.2 o20 Goh vq! ff) ajire 6 rhL Copy and translation of written amendment) Submission (Article 134-8 of the Patent Law) February 26, 1993 prisoner

Claims (1)

[Claims] 1) Having human ZP3 activity or at least partially human ZP3 immunogenicity A pure polypeptide or a functional derivative thereof characterized by: 2) The polypeptide according to claim 1, characterized in that it is produced recombinantly. 3) The following array: [There is an array] The polypeptide according to claim 1 or 2, characterized in that it contains at least a part of Do. 4) Claims 1 to 3, characterized in that at least a portion thereof is glycosylated. Polypeptide according to any one of the above. 5) The following array: [There is an array], [There is an array], [There is an array], [There is an array], [There is an array], or [There is an array] The polypeptide according to claim 3 or 4, characterized in that it contains at least one of Do. 6) The following array: [There is an array] Human ZP3 activity or at least a portion of A polypeptide having partial human ZP3 immunogenicity. 7) Coating at least a part of the polypeptide according to any one of claims 1 to 6. Nucleic acid sequence to be coded. 8) The following array: [There is an array] or a functional derivative thereof. Nucleic acid sequences on the website. 9) A vector comprising the nucleic acid sequence according to claim 7 or 8. 10) A host comprising the nucleic acid sequence according to claim 9 or the sequence according to claim 7 or 8. Principal cell. 11) A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 6. 12) A polypeptide comprising the polypeptide according to any one of claims 1 to 6 and an adjuvant. immunocontraceptive vaccine. 13) Contains an antibody that recognizes the polypeptide according to any one of claims 1 to 6. Immunocontraceptive vaccine. 14) A nucleic acid sequence encoding the polypeptide according to any one of claims 1 to 4. transform a host cell with a vector containing the vector, and culture the host cell in a suitable medium. and then isolating the polypeptide from the culture. A method for producing the polypeptide according to item 1. 15) Adjuva and the polypeptide according to any one of claims 1 to 6 to a suitable animal. The B-lymphocytes of said animal are harvested after a suitable period of time and the antibody-producing cells are cells can be selected and immortalized, and then they can be cultured and antibodies can be isolated from the cultures. A method for producing an antibody according to any one of claims 1 to 6, characterized in that: