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人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子--半透明带结合

宋力雯, 汪玉宝, 倪崖, 何亚萍, 洪爱真, Hinsch E, Hinsch K D, 周思畅, 袁玉英, 石其贤, 徐万祥

复旦大学上海医学院.上海 200032；上海市计划生育科学研究所.上海 200032；浙江省医学科学院.浙江，杭州 310013；皮肤与男科学中心，Juetus Liebig 大学.吉森 35392；支薪雇员公共机构联邦保险Riff康复门诊部.Borkum 26757

摘要

分析大肠杆菌表达的重组人卵透明带--3(r--huZP3)蛋白两个肽段(r--huZP3a~(22---176)及r--huZP3b~(177---348))的免疫原性，比较两者抗血清体外抑制人精子-半透明带结合的能力。以制备性聚丙烯酰胺凝胶电泳(PAGE)纯化的大肠杆菌表达蛋白为抗原，主动免疫新西兰兔产生抗r--huZP3a~(22---176)及huZP3b~(177---348)抗血清；通过ELISA测定比较两者的抗体应答水平；以蛋白印迹和免疫组化方法测定两者抗血清同重组表达蛋白、天然人卵ZP以及卵巢组织的反应性；通过竞争性半透明带结合试验(hemizona assay, HZA)观察两者抗血清体外抑制人精子-ZP结合能力。 结果显示：未与大分子蛋白载体耦联的r--huZP3a~(22---176)和r--huZP3b~(177---348)抗原都在免疫兔中产生了较高的抗体滴度，而且它们的抗血清可识别大肠杆菌表达的各自重组ZP3肽以及人卵细胞表面天然ZP，两者抗血清也都能在体外抑制人精子--ZP结合。由此可见，r--huZP3a~(22---176)及r--huZP3b~(177---348)蛋白具有良好免疫原性，所产生抗血清也都显示出细胞和组织特异性。因此，单一或合并两个huZP3肽段均可作为抗原研制检 测不明原因性不孕妇女中是否存在抗自身ZP抗体的诊断试剂盒，另外它们的抗血清也能用于鉴定已知huZP3表位肽段的最小B--细胞表位基序。

关键词： 人卵透明带; 重组ZP3 肽; 抗血清; 卵母细胞; 半透明带结合试验

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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宋力雯, 汪玉宝, 倪崖, 何亚萍, 洪爱真, Hinsch E, Hinsch K D, 周思畅, 袁玉英, 石其贤, 徐万祥. 人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子--半透明带结合[J]. 生理学报 2005; 57 (6): .

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).