目的：建立胱抑素 C 蛋白（Cys C）原核表达系统，表达、纯化并鉴定重组的人 Cys C 。方法在不改变氨基酸序列的前提下，对 Cys C 的编码基因进行大肠杆菌同义密码子偏好性优化后，人工合成其序列，PCR 扩增Cys C 基因，在原核系统中表达重组人 Cys C 。纯化表达的 Cys C 重组蛋白，采用间接 ELISA 法测定其反应灵敏度及免疫原性。结果十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定可知，重组基因在大肠杆菌中获得了有效表达，相对分子质量约为35×103，Western blot 结果进一步说明重组蛋白为 Cys C 蛋白，间接 ELISA 结果显示，在包被浓度为0．5 mg／L 时便产生了阳性值。结论成功建立了 Cys C 原核表达系统，Cys C 蛋白作为抗原具有良好的免疫反应性与免疫原性，为制备相应的抗原诊断单克隆抗体打下了基础，同时也为开发 Cys C 快速诊断ELISA 试剂盒提供了可选原料。%Objective To establish a prokaryotic expression system of cystatin C (Cys C) protein for express-ing ,purifying and identifying recombinant human Cys C protein .Methods Under the premise of unchanging amino acid sequence ,the Cys C coding gene was performed the Escherichia(E .) coli synonym codon bias optimization and its sequence was artificially synthesized .The Cys C gene was amplified by PCR .The human recombinant Cys C pro-tein was expressed in the prokaryotic system .The expressed Cys C recombinant protein was purified .Its reaction sen-sitivity and immunogenicity were assayed by indirect ELISA .Results The SDS-PAGE identification indicated the re-combinant gene gained the effective expression in E .coli ,the molecular weight of recombinant Cys C protein was 35 × 103 .The Western blot results further revealed that the recombinant protein was Cys C protein ,the indirect ELISA results displayed that when the coating concentration was 0 .5 mg/L ,the positive value would be produced .Conclusion The pro-karyotic expression system is successfully established ,Cys C protein as antigen has better immunoreactivity and im-munogenicity ,which lays a foundation for preparing corresponding antigen diagnostic monoclonal antibody ,meanwhile also provides the optional raw material for developing Cys C rapid diagnosis ELISA reagent kit .
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