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Gene Cloning of a Porcine Zona Antigen Which Cross-Reacts With Human Zona Pellucida

Research Project

Project/Area Number 01570944
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Obstetrics and gynecology
Research InstitutionHyogo College of Medicine

Principal Investigator

KOYAMA Koji  Hyogo College of Medicine, Associate Professor,, 医学部, 助教授 (00068496)

Co-Investigator(Kenkyū-buntansha) YAMASAKI Noriyuki  Hyogo College of Medicine, Instructor, 医学部, 助手 (50174644)
Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1989: ¥1,300,000 (Direct Cost: ¥1,300,000)
KeywordsZona Pellucida / Inhibition of Fertilization / Contraceptive Vaccine / Gene Cloning / Monoclonal Antibody / 単クロ-ン抗体 / 交叉抗原
Research Abstract

Five monoclonal antibodies (Mads), 2A1, 2G3, 4A2, 4E12 and 5H4, were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zona pellucida (s-PZP). Each Mab reacted not only with s-PZP, but also reacted with deglycosylated s-PZP by trifluoromethanesulfonic acid (TFMS) treatment. They also reacted with intact ZP of porcine and human oocytes. Three (4A4, 4E12 and 5H4) of the 5 Mabs showed a significant blocking effect on human sperm binding and penetration to human ZP. The 5H4 showed a strong reaction with ZP4 and ZP1 glycoprotein families, while other 4 Mabs reacted more strongly with ZP3 than ZP4. The reactivity of 5H4 with s-PZP was destroyed by chymotrypsin digestion but the antigen epitope was resistant to proteolyses by trypsin and lysylendopeptidase. A peptide fragment reactive to 5H4 was isolated by a reverse-phase HPLC of lysylendoproteinase-treated ZP4 glycoproteins and the molecular weight was determined to be 7kd by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 would be a good candidate for development of contraceptive vaccine.
To isolate a cDNA for ZP4, we constructed a cDNA library of a porcine ovary, and antibody screening was done by using 5H4. The three clones that reacted with 5H4 were isolated and their nucleotide sequence determined. One of them showed extreme homology with a gene for collagen and the other two clones showed no homology with known sequences. However none of them contained the nucleotide sequence for ZP4 determined by the amino acid sequencing. We screened the cDNA library of a human ovary and isolated 2 clones that reacted with 5H4, but the deduced amino acid sequence did not show any homology with porcine ZP4.

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] 長谷川 昭子、香山 浩二、礒島 晋三: "ヒト受精に関係するブタ透明帯構成蛋白質の解析" 日本受精着床学会雑誌. 7. 192-195 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Akiko Hasegawa,Koji Koyama,Shinzo Isojima(ed.by Y.Tomoda,Y.Yagami): "Effect of antisera to four glycoprotein families(Zp_1,ZP_2,ZP_3,ZP_4)of porcine zona pellucida on human spermーZP interaction and active immunization of hamster with ZP_4." In Proceedings of the 4th Annual Meeting of Japan Society for Basic Reproductive Immunology. 47-50 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Akiko Hasegawa,Koji Koyama,Shinzo Isojima: "Isolation of four major glycoprotein families(ZP_1,ZP_2,ZP_3,ZP_4)of porcine zona pellucida and characterization of antisera raised to each glycoprotein family." Acta Obst.Gynaec.Jpn.43. 221-226 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Akiko Hasegawa, Koji Koyama, Shinzo Isojima: "Analysis of porcine zona pellucida glycoproteins related to human in vitro fertilization" J. of Fertilization and Implantation. 7. 192-195 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Akiko Hasegawa, Koji Koyama, Shinzo Isojima: "Effect of antisera to four glycoprotein families (ZP_1, ZP_2, ZP_3, ZP_4) of porcine zona pellucida on human sperm-zp interaction and active immunization of hamster with ZP_4" Proceedings of the 4th Annual Meeting of Japan society for Basic Reproductive Immunology (ed. by Y. Tomoda, Y. Yagami). 47-50 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Akiko Hasegawa, Koji Koyama, Shinzo Isojima: "Isolation of four major glycoprotein families (ZP_1, ZP_2, ZP_3, ZP_4) of porcine zona pellucida and characterization of antisera raiseds to each glycoprotein family." Acta Obstetrics and Gynecology Japan. 43. 221-226 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 長谷川 昭子,香山 浩二,礒島 晋三: "ヒト受精に関係するブタ透明帯構成蛋白質の解析" 日本受精着床学会雑誌. 7. 192-195 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Akiko Hasegawa Koji Koyama Shinzo Isojima: "Isolation of four major glycoprotein families (ZP_1,ZP_2,ZP_3,ZP_4) of porcine zona pellucida and characterization of antisera raised to each glycoprotein family" Acta Obstetrics and Gynaecology Japan. 43. 221-226 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] Akiko Hasegawa et.al.: "Immuroogenicity of a 92KDa component of porcine zona pellucida isolated using a monoclonal antibody(3A4-2G1)exclusively cross-reactive with porcine and buman zona pellucidae" J.Reprod.Immunol.14. 73-82 (1988)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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