WO1995008569A1 - NOUVELLE PROTEINE sp38 ET VACCIN CONTRACEPTIF - Google Patents

NOUVELLE PROTEINE sp38 ET VACCIN CONTRACEPTIF Download PDF

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Publication number
WO1995008569A1
WO1995008569A1 PCT/JP1994/001569 JP9401569W WO9508569A1 WO 1995008569 A1 WO1995008569 A1 WO 1995008569A1 JP 9401569 W JP9401569 W JP 9401569W WO 9508569 A1 WO9508569 A1 WO 9508569A1
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Prior art keywords
protein
sperm
peptide
leu
ser
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PCT/JP1994/001569
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English (en)
Japanese (ja)
Inventor
Tsuneatsu Mori
Etsuko Mori
Tadashi Baba
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Kirin Beer Kabushiki Kaisha
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • 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

Definitions

  • the present invention relates to a novel protein (sp38) and a novel protein having antigenicity in a human or animal used for a contraceptive vaccine for the human or animal. It relates to the peptide fragment and its manufacturing method. In addition, the present invention relates to antibodies against such antigens and the background of their use as contraceptives.
  • contraceptive methods include pills, IUDs (intrauterine devices), condoms, sterile surgery, and oral administration of low-molecular-weight synthetic substances that reduce sperm count and motility.
  • IUDs intrauterine devices
  • condoms condoms
  • sterile surgery sterile surgery
  • oral administration of low-molecular-weight synthetic substances that reduce sperm count and motility.
  • immunological contraceptive methods have been attempted, and for example, methods for preventing implantation of a fertilized egg by using, for example, hCG vaccine are known. .
  • the method of blocking fertilization at an earlier stage is considered to be more desirable and more desirable, as is the ability to prevent the fertilization of the egg zona pellucida and sperm. Fertilization can be prevented by using white matter as a contraceptive vaccine or by administering antibodies to them. The following is a brief description of the ways in which stopping and achieving contraceptive goals have been attempted.
  • Fertilization of mammals is based on the fact that eggs that have matured and ovulated in the ovaries encounter fertilized sperm as they pass through the female genital tract, and that they meet in a large part of the fallopian tubes. It is started. Outside the egg cell membrane, there is a glycoprotein layer called the zonape 11 uci da (ZP), which is the extracellular matrix of the egg. It must specifically bind to and penetrate this ZP before it can fuse with the egg cell membrane. In some species, including humans, only spermatozoa that have undergone a so-called acrosome reaction (acr0s0 mereacacton) bind and penetrate ZP.
  • acrosome reaction acr0s0 mereacacton
  • This reaction is the fusion of the outer membrane of the acrosome of the sperm frontal region with the cell membrane, which is detached, and in these species, it is exposed after this acrosome reaction. It is thought that the sperm antigen plays a leading role in binding to the zona pellucida. Therefore, it is thought that an appropriate contraceptive effect can be expected by efficiently inducing an antibody against such a sperm antigen having ZP binding properties.
  • broacrosin As a protein that is exposed to the sperm surface after the acrosome reaction and has a ZP binding activity, broacrosin (proacrosin) in butter sperm is known.
  • Proacrosin is a zymogen of aosin, a type of serine protease. This molecule is composed of a serine protease group that is abundant in other organs such as trypsin and a homolog of 30 to 40% of amino acids. And produce sperm-specific antibodies There is a problem as an immunogen for this.
  • contraceptive vaccines may include ZP4, a glycoprotein that is translucent to the human egg, and ZP0 that originates from the Hamster oviduct. Yes. However, none of these are qualified antigens because their molecular structures in humans are unknown. A vaccine method using the human sperm acrosome protein sp10 as an antigen has also been considered, but since the physiological function of sp10 is completely unknown, its mechanism of action is considered. Is not clear.
  • sperm immobilization antibodies are also being considered as an immunological contraceptive method.
  • the drawbacks are that the corresponding antigen is unknown, or cannot be used as an active immunogen that lacks specificity and can be expected to have a long-term effect. There is.
  • the present invention relates to a peptide and a code as a novel antigen having a clear mechanism of action, which can be used as a contraceptive vaccine.
  • the purpose is to provide a DNA sequence that can be
  • the present invention provides a contraceptive vaccine comprising all or a part of the peptide by knowing the sequence of such peptide. The purpose is to provide it.
  • the present invention has an object to provide a contraceptive containing an antibody against the peptide.
  • novel protein s ⁇ 3 provided by the present invention is provided.
  • the first of 8 is sP38 derived from a puta having the sequence shown in FIG. 1 (hereinafter referred to as buta s ⁇ 38).
  • the second of the novel proteins provided by the present invention is human-derived sp38 having the sequence shown in FIG. 3 (hereinafter referred to as human sp38). ).
  • the D ⁇ sequence according to the present invention encodes all or a part of the butter s ⁇ 38 or the human sp38.
  • the contraceptive vaccine according to the present invention is a whole or a partial peptide of the above-mentioned novel protein according to the present invention, and has a binding activity to a ZP protein. Or a substance having an antigenicity capable of producing an antibody that inhibits the binding of sperm to ZP protein in vivo.
  • the contraceptive according to the present invention is an antibody against all of the novel proteins according to the present invention or a partial peptide thereof, and has a binding activity to a ZP protein. And an antibody against an antigenic portion capable of producing an antibody that inhibits the roll of Z or sperm with ZP protein in vivo. .
  • FIG. 1 shows the amino acid sequence of buta sp38.
  • FIG. 2 shows a DNA sequence encoding puta sp38, which includes a coding region of No. 15—1067.
  • FIG. 3 shows the amino acid sequence of human sp38.
  • FIG. 4 shows a DNA sequence encoding human sp38, which includes a coding region of 37-1092.
  • Figure 5 shows the HP of the protein component of the spermatozoa in the epididymis of the pigeon.
  • FIG. 1 shows the results of LC analysis and the ZP binding activity of the fraction protein.
  • Figure 6 shows the results of reversed-phase HPLC analysis of the fractions having ZP binding activity and the ZP binding activity of the fractions, showing two distinct peaks in ZP binding activity. Marks a and b were observed.
  • FIG. 7 shows the results of SDS polyacrylamide electrophoresis (SDS-PAGE) of elution fraction numbers 14 to 24 in FIG.
  • FIG. 8 shows the results of Western blotting analysis, in which lanes 1 and 2: the ZP-binding protein fraction (peak b) and the ZP protein were compared. Results, lane 3: results from fractions of fraction 15 (55 kDa and 53 kDa proteins) and anti-human sperm broacrosin antibody. No. 4: The results are for the ZP-binding protein fraction (peak b) and anti-sperm sperm broacrosin antibody.
  • FIG. 9 is a diagram showing the results of reverse phase HPLC analysis of the peptide fragment obtained by enzymatic digestion of sp38, and the amino acid sequence of the peptide fragment.
  • Figure 10 shows the separation of the piotinylated ZP protein by reverse-phase HPLC into 9 OkDa, 55 kDaoi, and 55 kDaS components.
  • FIG. 7 shows the results, showing that a fraction having a binding activity to both sp38 and broacrosin was obtained.
  • Fig. 11 shows the results of SDS-PAGE of elution fraction numbers 19 to 24 in Fig. 10, where sp38 and the protein mouth were all 90 kD. This suggests that it binds to the same binding site of ZP protein in a.
  • Fig. 12 shows the results of an experiment on the competitive inhibition of broacrosin and sp38. Hataichi is on sp38, and ⁇ is the brower. About crossin.
  • Figure 13 shows the SDS-PAGE results of the N-glycanase digest of sp38, lane 1 after digestion and lane 2 before digestion.
  • Figure 14 shows the results of SDS-PAGE of the fusion proteins of sp38 and T7 genel 0 (lane 1: CBB staining), and the biotinylated ZP protein.
  • Fig. 15 shows the results of a Stan Blot analysis (lane 2) using the probe as the probe quality.
  • Fig. 15 shows the results obtained using the buta sp38 fusion protein antibody. The results of Stanplot analysis are shown.
  • Lane 1 Purified sp 38, lane 2: buta sperm cells; lane 3: buta s. E. coli expressing a fusion protein of P38 and T7 gene 10; lane 4: human Escherichia coli expressing the fusion protein of sp38 and GST.
  • Lane 5 Escherichia coli as a control.
  • Fig. 16 shows the results of Northern blot analysis, where lanes 1 to 4 are poly (A) derived from testis, liver, kidney, or spleen, respectively, of submucosa. ) + RNA.
  • FIG. 17 shows the results of immunostaining of sperm cells.
  • A sperm before induction of acrosome reaction
  • B sperm after induction of acrosome reaction
  • C metabolism before induction of acrosome reaction.
  • FIG. 18 is a diagram showing a decrease in ZP binding activity upon maturation of broacrosin to acrosin.
  • FIG. 19 shows the inhibition of the binding between sp38 of the synthetic peptide having the partial amino acid sequence shown in Table 1 and the ZP protein. Indicates the case of a partial peptide of 5p38, and ⁇ - ⁇ indicates the case of a partial peptide of broacrosin.
  • Figure 20 shows the inhibition of sperm-egg binding by the antibody.
  • A The appearance of the egg cell in the presence of non-sensitized IgG.
  • B Egg cells in the presence of anti-sp38 fusion protein antibody. In this case, sperm cell binding is not observed.
  • FIG. 21 shows the results of SDS-PAGE of transformant DH5, wherein lane 1 indicates before expression and lane 2 indicates after expression.
  • B. sp. 38 provided by the present invention was isolated from sperm collected from the epididymis of the B. aureus by using the ZP binding ability as an index. Protein. This puta sP38 is a protein that is not expressed before the induction of the acrosome reaction, but is exposed on the surface of the sperm after the acrosome reaction. It is thought that the sperm pass through the ZP and become fertilized.
  • This protein has a specific binding activity to the ZP protein, as is evident from the examples described later, and it is competitive with procyrosin. .
  • the antibody against B. sp. 38 acts specifically on sperm, inhibiting the binding of sperm to ZP protein and aggregating sperm. Has been confirmed.
  • a human sp38 is obtained by obtaining a probe and a probe by referring to the sequence of the human sp38, and using the probe to obtain a human testis cDNA library. The protein was confirmed to be present by analysis of the obtained cDNA expression product.
  • human sp38 is also considered to be a protein exposed on the sperm surface after the acrosome reaction, similar to the butter sp38, and this sp38 is responsible for the binding of sperm to ZP. Later, the sperm may add ZP and lead to fertilization. Therefore, human sp38 also has a specific binding activity to the ZP protein, similar to bushu sp38, and an antibody against it specifically acts on sperm. To inhibit the binding of sperm to ZP protein. Therefore, human sp38 also has a use as a contraceptive vaccine in the same way as the above-mentioned buttersp38, and the contraceptive effect is also obtained with respect to antibodies against it. You can expect it.
  • peptides comprising part of the sequence shown in FIG. 3 are also included in the scope of the present invention.
  • the portion of the sequence shown in FIG. 1 which has at least the sequence from No. 253 to No. 269 is shown in FIG.
  • the peptide inhibits the binding of sp38 to the ZP protein, and also binds to this partial peptide.
  • Antibodies to it inhibit sperm-egg binding. While not being bound by the following theory, as shown below, this sub-peptide, along with no. You can see the sequences No. 365 to No. 372 of the cinnamon and the homologue, and for the human sp38 the homologue with these sequences. Is the O from number 257 to number 267
  • a contraceptive effect can be expected even with an antibody obtained using these partial peptides as antigens.
  • amino acids may be used as long as they retain the binding activity to the ZP protein or an antigen capable of producing an antibody that inhibits the binding of sperm to the ZP protein in vivo.
  • an antigen capable of producing an antibody that inhibits the binding of sperm to the ZP protein in vivo buta s ⁇ 38 and human sp38 in which acid addition, insertion, deletion, deletion or substitution has occurred are also included in the scope of the present invention.
  • a DNA encoding a butter sp 38 and a human sp 38 there is further provided a DNA encoding a butter sp 38 and a human sp 38.
  • Typical sequences of DNAs encoding these proteins are shown in FIGS. 2 and 4, respectively, which have all or part of the base sequence.
  • nucleotide sequence encoding it can be easily determined, and as a result, shown in FIG. 1 or FIG.
  • Various nucleotide sequences encoding all or a part of the sequence to be obtained can be appropriately selected. Therefore, the nucleotide sequence encoding the whole or a part of the sequence shown in FIG. 1 or FIG. 3 according to the present invention corresponds to the nucleotide sequence shown in FIG. Is identical to all or part of the nucleotide sequence shown in FIG. 4 except that codons that have a degenerate relationship are used. Thus, sequences that encode the same peptide are also meant.
  • the DNA according to the present invention is synthesized using a part derived from a natural product, a whole synthesized product, or a product derived from a natural product. It may be something (ie, semi-synthetic).
  • One of the typical methods for obtaining such DNA is to cut out the gene from a genomic library derived from butter or human sperm. .
  • this method a method commonly used in the field of genetic engineering, for example, from a genomic library derived from puta or human sperm, for example, There is a method of screening using a suitable probe. Specific examples of this method will be described in detail in Examples described later.
  • the DNA sequence according to the present invention is contained in a state capable of replicating in a host cell and expressing a protein encoded by the DNA sequence.
  • One expression vector is provided.
  • a host cell transformed with the expression vector is provided, and the host-vector system is not particularly limited.
  • a white matter expression system can be used.
  • the procedures and methods for constructing the vector according to the present invention can use those commonly used in the field of genetic engineering.
  • the expression vector according to the present invention is based on the above-described present invention in order to actually introduce the vector into a host cell to express the desired protein.
  • the DNA that controls its expression that is, the transcriptional motor, such as the mouth motor, the transcription initiation signal, the ribosome binding site, the translation termination signal, and the transcription termination signal It is preferable to have a control signal and a translation preparation signal. These may be present in the expression vector according to a conventional method.
  • the method of transforming a host cell by this vector can also use a method commonly used in this field.
  • the above transformant can be obtained by culturing the transformant in a suitable medium and isolating the above-mentioned protein according to the present invention from the culture. Therefore, according to another aspect of the present invention, there is provided a method for producing a novel protein according to the present invention.
  • the cultivation of the transformant and its conditions may be essentially the same as those for the host used.
  • recovery and purification of the novel protein from the culture solution according to the present invention can be carried out according to a standard method.o
  • the antibodies against buta sp 38 and human sp 38 inhibit the binding of sperm to ZP protein.
  • a pharmaceutically acceptable carrier comprising all or part of the peptide shown in FIG. 1 or FIG. 3, and a pharmaceutically acceptable carrier.
  • Contraceptive vaccines are provided.
  • a pharmaceutically acceptable carrier comprising all or a part of the peptide shown in FIG. 1 or 3 as an antigen.
  • the protein according to the present invention as an active ingredient of a contraceptive vaccine is a human sp38 or a partial peptide thereof. It is preferable that the antibody retains antigenicity capable of producing an antibody in vivo that inhibits the binding of sperm to ZP protein.
  • the contraceptive vaccine according to the present invention can be administered to humans via either the oral or non-oral route (eg, intravenous, intramuscular, subcutaneous, transdermal). And can be administered to animals other than humans. Accordingly, the contraceptive vaccine according to the present invention may contain, depending on its administration route, a suitable pharmaceutically acceptable carrier and, if desired, a stabilizer and a preservative. In any case, the dosage form is appropriate.
  • the dose and frequency of the contraceptive vaccine are ultimately determined by the guidance of a specialist, but once a dose of 1 to 1 mg of protein as an active ingredient from 1 to 1 mg of protein. It is possible to administer several doses at 2-week or 4-week intervals.
  • the antibody against human sp38 or a partial peptide thereof is used. It is preferable that the antibody be an antibody against a substance that retains antigenicity and can produce an antibody that inhibits the binding of sperm to ZP protein in vivo.
  • An antibody as an active ingredient is obtained by immunizing an animal.
  • Antisera, polyclonal antibodies obtained by purifying them, and monoclonal antibodies can be used. Local antibodies are preferred.
  • Monoclonal antibodies can be obtained according to methods commonly used in this field.
  • the contraceptives according to the present invention can be administered to humans via any of the routes of administration, parenteral and parenteral (eg, intravenous, intramuscular, subcutaneous, rectal, transdermal). And can be administered to animals other than humans. Accordingly, the contraceptives according to the present invention may contain, depending on the route of administration, a suitable pharmaceutically acceptable carrier and, if desired, a stabilizer, a preservative and the like. Both are in a suitable dosage form.
  • the amount of the antibody as an active ingredient in the contraceptive pill is ultimately determined by the guidance of a specialist, but a sufficient antibody titer can be obtained in the active area (tubal tube). It is preferred that the amount be in the range without side effects.
  • Protein was extracted from the spermatozoa of the epididymal tail (2.5 ml) using a buffer containing a data agent (1). This extract was subjected to gel filtration according to the method for purifying acrocin (2), and the eluted fraction was subjected to solid-phase binding assay. Was used to evaluate the ZP binding activity. A protein fraction having ZP binding activity (1 O mg of protein) was recovered and dialyzed against 7 M urea solution adjusted to pH 3 with HC1.
  • the dialysis solution was pre-equilibrated with 7 M urea, 0.2 M NaCl, and pH 3 cation exchange HPLC column TSK ge 1 SP-5PW ( 7.5 x 75 mm: Add to Tohoku Sodium), and let it stand at room temperature for 200 minutes with a linear gradient of 0.2 to 1 M NaCl in PH3, 7 M urea. It eluted in each fraction lml at a flow rate of 0. ⁇ ⁇ ⁇ (Fig. 5).
  • the “method for measuring ZP binding activity” using a solid phase binding assay is as follows.
  • ZP glycoprotein was prepared from ovarian follicular ova and piotinylated according to a conventional method (3) (1).
  • the eluted sperm derived from ⁇ white each off La click tion of, 0 1 MN a HC 0 3 your good beauty 2 m M p-aminobenzami di ne . - the presence of (p AB), ELISA-flops over door We coated each of the wings.
  • Wash buffer (1 0 m MT ris -. HC 1, p H 7 4, 0 1 5 MN a C l, 2 m MC a C l 2, 2 m M p -. AB, 0 0 5% T ween - 20)
  • the peptide fragments were eluted using a concentration gradient separation method under the following conditions for 40 minutes, 2 to 50% solvent B, and a flow rate of 0.25 ml Z.
  • solvent A 0.05% Trifnoreo mouth acetic acid aqueous solution
  • solvent 0 ⁇ 0 2%
  • the amino acid sequence was analyzed using a gas-phase sequencer (A ⁇ 1 iedBiosystemsmodel470A). The identity of the resulting PTH-amino acids was determined by reverse phase HPLC of these peptides, performed using homogeneous HPLC, as shown in Figure 9. It is as shown. AP-1 to AP-6 of the peptide fragment and the amino acid sequence at the N-terminus were also as shown in FIG. The amino acid sequence of AP-3 was consistent with the amino acid sequence at the N-terminus. Also, eight? It was found that the ⁇ terminus of ⁇ 3 and the N terminus of AP-4 were continuous.
  • amino acid sequence of each of these peptides is also found in the amino acid sequence of the Nationala 1 Biochemical Research Foundation NBRF) ⁇ Homologous to known amino acid sequences in One was not seen.
  • Puta's ZP protein consists of three sulfated glycoproteins, 90 K
  • Example 4 1-1 Construction of a cDNA library, screening and sequencing
  • testicle po 1 y (A) + RNA should be treated with 10 mM of methy l me rcury hy dr ox ide. Except for denaturation for one minute, use E. coli Y as the host according to Pharmacia LKB Biotechno 1 ogy ⁇ manu factures, protocol (Pharmacia LKB Biotechno 1 ogy). Double strand cDNA was prepared using Igt11 as a vector, and a library was constructed. A plaque hybridization method using a mixture of oligonucleotides corresponding to the amino acid sequence KVYVMLHQK as the probe
  • Oligonucleotides encoding the amino acid sequences of K71 (Lys) and K79 (Lys) analyzed by peptide mapping Northern blot analysis was carried out on po1y (A) + RNA of the testis posibuta, using a mixture of these as a probe.
  • A po1y + RNA of the testis posibuta
  • a full-length messenger including the complete open-ended room, is approximately 1400 bases I knew it was a pair.
  • Analysis of the hydrophobic region using the Kyteand Doo 1 itt 1e method showed that only the N-terminal region (amino acid residues 21 to 37) had high hydrophobicity. I knew it was an area.
  • the most likely cleavage site is between A39 (Ala) and P40 (Pro). Met.
  • the molecular weight calculated from the amino acid sequence inferred from the cDNA was 39 KDa, and the molecular weight excluding the signal sequence was 35 KDa.
  • the results of amino acid sequence analysis at the N-terminal started from S52 (Ser).
  • the molecular weight of the polypeptide from S52 (Ser) to L350 (Leu) was calculated to be 34 KDa.
  • sp38 derived from epididymis is cleaved at the N-linked sugar chain with N-dalicanase and subjected to SDS-PAGE (molecular weight: 34 KDa, Fig. 13). Is consistent with. N113 (A sn), N186 (A sn) and N 339 (A sn) had sites capable of binding asparagine-linked sugar chains. No known amino acid sequences in NBRF and no significant homologues for sp38 were found.
  • Example 4-2 ⁇ Expression of sp38 cDNA in E. coli and Preparation of Antibody against Expressed sp38 Fusion Protein S65 (Ser) of sp38 Protein The Sau11!
  • Escherichia coli was transformed with the plasmid from which PSD38 was obtained, and the expression was induced by IPTG.
  • the binding between the expressed fusion protein of sp38 and T7 gene 10 and ZP was determined by Western blot analysis using biotinylated ZP as a probe.
  • Figure 14 Polyclonal antibody against sp38 fusion protein purified by SDS-PAGE was immunized with Freunds com leteadju Vant (Gibc0.NY) using N ew Zeal and white ⁇ After purifying the IgG fraction prepared from egrets with Protein G Sepharose-Scalam, the antigen-specific antibody was further purified. (10) o Purified using sP38 antigen, a pig sperm immobilized on PVDF membrane
  • the antibody obtained in this way reacts with sP38 purified from epididymal sperm (Fig. 15; lane 1). This confirmed that sp38 (a fusion protein thereof) was expressed in E. coli 0
  • Tissue RNA from testis, liver, kidney, and spleen from oocytes, and organs using Ordoxy T-column (Pharmacia LKB Biotee hnology) was prepared from po 1 y (A) + RA. After denaturation (10) of p0y (A) + RNA with glyoxal, the mixture was electrophoresed on a 1.2% agarose gel and transferred to a GeneScrenePlus TM membrane. Hybridization (7) was carried out using the same oligonucleotide mixture as the probe.
  • Osuvian evening sperm recovered from the epididymis were capacitated (12).
  • Some spermatozoa may have a concentration of 5 / M in the canesome mediophone (A23187: Sima) to induce an acrosome reaction.
  • A23187: Sima canesome mediophone
  • These sperm were washed with PBS and sperm Apply the solution 1% over 20 minutes.
  • the cells were fixed with PBS containing la forum aldehyde. Overlay the sperm suspension in 0.5 ml FCS and centrifuge. I did it. After washing the sperm spun down in FCS several times with PBS, use 2% FCS in PBS to avoid non-specific staining 4.
  • the partial amino acid sequence sp 38 shows that the amino acid sequence at positions 256 to 2666 of sp38 Corresponding KRLSKAKNL, IE (residues K256-E2666) and KRLQQLIE corresponding to the amino acid sequence of amino acids from 365 to 3272 of broacrosin (366-5 — £ 372 residues).
  • K365 (Lys) and R366 (Arg) in the base sequence of this broacrosin is from the protein mouth. It has been reported that it is cut off during machination of acrocin (7).
  • Example 6-2 Each synthesis of sp38, broacrosin, solid-phase binding assay of peptide fragment
  • Each synthetic peptide fragment having a partial amino acid sequence of sp38 and broacrosin shown in Table 1 is the system added to the C-terminal.
  • the residue was linked to tyroglobulin derived from human via a residue.
  • Rabbits were immunized with this using Freund scom leteadj uvant.
  • the increase in the antibody titer was confirmed by ELISA using each of the synthetic peptides shown in Table 1 adsorbed to a highly adsorptive ELISA plate. Purification of IgG fraction is performed by Protein G Sepharose column.
  • specific antibodies were further purified using the puta sperm sp38 antigen immobilized on the PVDF membrane or the pro-oral cinnamate (10).
  • Anti-buta-sp38 fusion protein purified specifically for each antigen-egret serum (Antibody I), peptide peptide having the partial amino acid sequence of sp38 shown in Table 1.
  • Ringer-bicarbone solution (m-KRB) gave fertilization ability (12).
  • puta ovary eggs were stored in a high salt storage solution (13), and desalted through several steps of m-KRB before use.
  • m - 3 0 / g Z m each antibody diluted 1 5 0 beta 1 a suspension of sperm gave fertility in KRB (5 X 1 0 6 or Z ml) 5 0 // 1: pressurizing e , 1 0 force Later, et 2 0 eggs, and the mixture was allowed to stand Te to 3 7 ° C 6 0 minutes in the C 0 2 Lee emissions queue base over data.
  • the sperm agglutination test used a micro ⁇ tray test method that was slightly modified from the report in (14). The fertilized ability was given to the sperm sperm collected from the tail of the epididymis, and the acrosome reaction was induced with a force receptor. We argued to be 0 5 / m 1.
  • the purified anti-sp38 fusion protein or rabbit antibody or non-sensitized IgG is diluted to various concentrations with PBS, and this solution 51 is added to the sperm suspension 11. It was baked at 37 ° C below the rough-finished film. After 60 minutes, the plate was observed with a phase contrast microscope to determine the presence or absence of aggregation. As shown in Table 3, the results are as shown in Table 3.
  • Cloning of human sP38 was based on the human testis that used Escherichia coli Y1090R as a host and sgt11 as a vector. c From DNA library (Clontech), sp. 38 c DNA cloning plasmid p N end of PSP38-13 The Ec0RI fragment ⁇ (1300bP) containing the translation region of was used as the probe, and the method was performed using the Park-Hyper-prediction method. . Hydration—Challenge at 60 ° C to homogenize positive clones, and add the human sp38c DNA insert to PUC19. Bronzing gave the plasmid pHSP38-4.
  • Example 9 Expression of 2 human sp38 cDNA in E. coli
  • Human sp38 cDNA was transformed into E. coli expression vector pGEX-13T (Pharmacia LKBB iotec hnology) using glutathione S-transferrer. It was expressed as a fusion protein with GST (GST).
  • GST GST
  • PCR was performed using TTTTGCTCC — 3 ′ and PHHSP 38 — 4 as a template.
  • Gene Amp trade name
  • PCRS system 9600 PERKINELMECET ⁇ S
  • 22 samples at 94 ° C for 1 minute, 58 ° C for 5 seconds, and 72 ° C for 2 minutes.
  • the test was performed under the conditions of the club.
  • the obtained PCR product was digested with BamHI and HindiII, inserted into the BamHI and HindiII sites of pGEX-3T, and the human sp38 and glutathione were digested.
  • An expression vector for the expression of a fusion protein with S-transferase was obtained.PGEXZHSP38 was obtained. E.
  • coli DH5 was transformed with the PGEXZHSP38, and the expression was expressed by IPTG. Guidance was provided. Transformant DH 5 S DS — PAGE No. The turn is shown in Figure 21. In FIG. 21, lane 1 is before expression induction, and lane 2 is after expression induction.
  • this fusion protein is confirmed by the fact that the antigen-specifically purified anti-puta sp38 fusion protein antibody shows cross-reactivity with the human sp38 fusion protein.
  • lanes 4 and 5 are obtained by solubilizing Escherichia coli expressing and not expressing the human sp38 fusion protein, and performing Western blot analysis. Only the human sp38 fusion protein shown in SEQ ID NO: 4 showed cross-reactivity with this antibody.
  • Leu Leu Gin lie Leu Ser Lys Leu Val Leu Asp Leu Ser Cys Glu Val
  • Organism name human sequence
  • Lys Leu Leu Gin lie Leu Ser Lys Leu Leu Leu Asp Leu Ser Cys Glu
  • Sequence type nucleic acid
  • Organism name human sequence

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  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Reproductive Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention se rapporte à un peptide comprenant la séquence illustrée dans la figure ou une partie de cette séquence. Ce peptide est une protéine s'exposant à la surface du sperme après la réaction des acrosomes, et l'anticorps contre ce peptide agit sur le sperme de façon spécifique pour empêcher la combinaison d'une cellule d'ovule avec le sperme. Ainsi, ce peptide peut servir de vaccin contraceptif.
PCT/JP1994/001569 1993-09-24 1994-09-22 NOUVELLE PROTEINE sp38 ET VACCIN CONTRACEPTIF WO1995008569A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP26168593A JP3537471B2 (ja) 1993-09-24 1993-09-24 新規蛋白質ｓｐ３８および避妊ワクチン
JP5/261685 1993-09-24

Publications (1)

Publication Number Publication Date
WO1995008569A1 true WO1995008569A1 (fr) 1995-03-30

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PCT/JP1994/001569 WO1995008569A1 (fr) 1993-09-24 1994-09-22 NOUVELLE PROTEINE sp38 ET VACCIN CONTRACEPTIF

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JP (1) JP3537471B2 (fr)
WO (1) WO1995008569A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1233026A1 (fr) * 1999-10-26 2002-08-21 Fuso Pharmaceutical Industries Ltd. Anticorps dirige contre les spermatozoides de la reaction postacrosomique du rat, et son utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03502571A (ja) * 1987-10-07 1991-06-13 ゾナゲン　インコーポレーテッド 不妊と避妊のための透明帯抗原と抗体の調製法及び使用
JPH05500654A (ja) * 1989-06-12 1993-02-12 アメリカ合衆国 クローン化透明帯遺伝子を基本とする避妊ワクチン

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03502571A (ja) * 1987-10-07 1991-06-13 ゾナゲン　インコーポレーテッド 不妊と避妊のための透明帯抗原と抗体の調製法及び使用
JPH05500654A (ja) * 1989-06-12 1993-02-12 アメリカ合衆国 クローン化透明帯遺伝子を基本とする避妊ワクチン

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1233026A1 (fr) * 1999-10-26 2002-08-21 Fuso Pharmaceutical Industries Ltd. Anticorps dirige contre les spermatozoides de la reaction postacrosomique du rat, et son utilisation
EP1233026A4 (fr) * 1999-10-26 2005-08-17 Fuso Pharmaceutical Ind Anticorps dirige contre les spermatozoides de la reaction postacrosomique du rat, et son utilisation
JP4610149B2 (ja) * 1999-10-26 2011-01-12 扶桑薬品工業株式会社 ラット先体反応後精子に対する抗体およびその利用

Also Published As

Publication number Publication date
JPH0789997A (ja) 1995-04-04
JP3537471B2 (ja) 2004-06-14

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