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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 immunogenicity of two peptides (r-huZP3a22-176 and r-huZP3b177-348) of recombinant human zona pellucida-3 (r-huZP3) protein expressed in Escherichia coli was analyzed. The antiserum against human sperm- Translucent band combined with the ability. Anti-huZP3a22-176 and huZP3b177-348 antisera were prepared by immunizing New Zealand rabbits with active protein of Escherichia coli purified by preparative polyacrylamide gel electrophoresis (PAGE). The level of antibody response was compared by ELISA. Western blotting and immunohistochemistry were used to measure the reactivity of the antisera with the recombinant protein and natural human ovary ZP and ovarian tissue. The antisera of the two groups were detected by competitive zona pellucida binding assay (HZA) -ZP binding ability. The results showed that both r-huZP3a22-176 and r-huZP3b177-348 antigens not coupled to the macromolecular protein carrier produced higher antibody titers in immunized rabbits and that their antisera recognized each of the E. coli expression Both recombinant ZP3 peptide and native ZP on the human egg cell surface, both antisera, also inhibit human sperm-ZP binding in vitro. Thus, the r-huZP3a22-176 and r-huZP3b177-348 proteins have good immunogenicity and the antisera produced also show both cell and tissue specificities. Therefore, single or combination of two huZP3 peptides can be used as antigens to develop diagnostic kits for detecting anti-ZP antibodies in unexplained infertile women. In addition, their antisera can also be used to identify known huZP3 epitope peptides Segment minimum B-cell epitope motif.