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Immunogenicity of recombinant human zona pellucida-3 peptides expressed in E. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding

Song Liwen, Wang Yubao, Ni Ya, He Yaping, Hong Aizhen, Elvira Hinsch, KlausDieter Hinsch, Choaw Sichang, Yuan Yuying, Shi Qixian, Xu Wanxiang

Shanghai Medical College Fudan University.Shanghai 200032；China；Zhejiang Academy of Medical Sciences.Hangzhou 310013,Zhejiang；Germany；Borkum Riff Rehabilitation Clinic of the federal insurance for salaried employees Institution (BfA).Borkum 26757

Abstract

The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides (r-huZP3a~(22--176) and r-huZP3b~(177--348)) expressed in E. coli through immunizing rabbits, and to evaluate the efficacy of their polyclonal antisera against r-huZP3a~(22--176) and r-huZP3b~(177--348) to inhibit in vitro human sperm-egg binding respectively. Male New Zealand rabbits were immunized using r-huZP3a~(22--176) or r-huZP3b~(177--348) as antigen respectively, which was purified through an improved method of preparative gel polyacryulamide gel electrophoresis. The antibody response level of r-huZP3a~(22--176) or r-huZP3b~(177--348) immunogen in rabbits was determined by ELISA using mouse ZP3-5 (amino acid sequence~(137--150) being completely conserved with huZP3~(138--151) sequence) and specific huZP3-14 (amino acid sequence~(327--340)) synthetic peptides as coating antigens respectively. The immunoreactivity and specificity of the anti-r-huZP3a~(22--176) and anti-r-huZP3b~(177--348) antisera with each r-huZP3 peptides, were tested by immunoblot and immunohistochemistry (using native huZP and human ovary section) respectively. A competitive hemizona assay (HZA) was used to evaluate the efficacy of the antisera against r-huZP3a~(22--176) and r-huZP3b~(177--348) to inhibit in vitro human sperm-egg binding. Both r-huZP3 peptides were able to induce higher antibody titers in rabbits. Each antiserum could specifically recognize or bind to each target r-huZP3 peptide expressed in E. coli and native human ZP in vitro. The antisera also inhibited sperm-egg binding in the HZA. These results show that r-huZP3a~(22--176) and r-huZP3b~(177--348) are of strong immunogenicity. They can be used to develop a kit for detecting whether there are autoantibodies to zona pellucida in unexplained infertile women, and their antisera might be useful tools for determining minimal B-cell epitope sequences of several known huZP3 epitope peptides.

Key words: zona pellucida, human;Recombinant ZP3 peptides;Immune serum;oocyte;Hemizona assay

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Citing This Article：

Song Liwen, Wang Yubao, Ni Ya, He Yaping, Hong Aizhen, Elvira Hinsch, KlausDieter Hinsch, Choaw Sichang, Yuan Yuying, Shi Qixian, Xu Wanxiang. Immunogenicity of recombinant human zona pellucida-3 peptides expressed in E. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding . Acta Physiol Sin 2005; 57 (6): (in Chinese with English abstract).