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

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

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-huZP3a22-176及r-huZP3b177-348)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huZP3a22-176及huZP3b177-348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-huZP3a22-176 和r-huZP3b177-348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-huZP3a22-176及r-huZP3b177-348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。

The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides （rhuZP3a^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.