1993 Fiscal Year Final Research Report Summary
Cloning of cDNA coding for zona pellucida protein and application of recombinant protein for contraceptive vaccine
| Project/Area Number |
04454427
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Hyogo Medical College |
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Principal Investigator |
AKATANI-(HASEGAWA) Akiko Hyogo Medical College, Obstetrics and Gynecology, Assistant, 医学部, 助手 (50212402)
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| Co-Investigator(Kenkyū-buntansha) |
KOMORI Shinji Hyogo Medical College, Obstetrics and Gynecology, Assistant, 医学部, 助手 (60195865)
KOYAMA Koji Hyogo Medical College, Obstetrics and Gynecology, Professor, 医学部, 教授 (00068496)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Porcine Zona Pellucida / Sperm Binding Inhibition / Fertilization Blocking / Contraceptive Vaccine / Recombinant Antigen / Porcine ZP1 Gene / Pet Vaccine / Zona Pellucida Epitope |
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| Research Abstract |
Porcine zona pellucida, which is cross-reactive to many animal species including humans, is separated into four glycoprotein families in two dimensional PAGE.Biochemical, immunochemical and molecular biological analyzes revealed that pZP1 is partially divided to pZP2 and pZP4, and that pZP3 contains two distinct glycoprotein families of pZP3 alpha and pZP3 beta. Essentially, the core proteins of glycoprotein families constituting porcine zona pellucida are three polypeptides of pZP1, pZP3 alpha and pZP3 beta.
Monoclonal antibody (MAb-5H4) produced in our laboratory reacted with pZP1 and pZP4, and could inhibit human sperm binding to zona pellucida. In order to determine MAb-5H4 epitope sequence, pZP4 was purified with preparative SDS-PAGE and subjected to Edman degradation. The entire sequence of pZP4 was determined by peptide mapping/cDNA cloning. A cloned cDNA coding for 198 amino acids corresponding to pZP4 plus NH_2-terminal region of pZP2. It is evident that pZP4 and pZP2 are derived from a common parent protein, that is pZP1. To determine the epitope of MAb-5H4, three peptides were synthesized by a solid method according to computer analysis. The epitope was determined to be constructed with ten amino acid sequence of Cys-Thr-Tyr-Val-Leu-Asp-Pro-Glu-Asn-Leu. The sequence may participate in the sperm-zona interaction.
The cloned cDNA was inserted into an expression vector to produce a recombinant protein [rpZP1(1-198)]. The protein was injected in rabbits to examine a contraceptive effect. The raised antibody reacted not only to rpZP1(1-198) protein but also to wild pZP4 and intact porcine zona pellucida. Moreover, the antiserum reacted with a rabbit zona pellucida, suggesting that rpZP1(1-198) protein has the ability to induce self-reactive antibodies. All of the four rabbits (*) immunized with rpZP1(1-198) did not concieve by mating with fertile-proven male rabbits. Thus, it is concluded that rpZP1(1-198) is effective as a contraceptive vaccine.
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