【摘要】 目的 获得具有免疫反应性的人胱蛋白酶抑制剂C(Cys C)重组蛋白。方法 从Hela细胞提取总RNA,采用RTPCR扩增获得人Cys C基因片段,与质粒pET30a连接构建重组表达载体,转化大肠埃希菌E.coli BL21(DE3)并加入IPTG诱导表达,表达产物经凝胶层析复性和亲和层析纯化后,用间接ELISA检测纯化后的Cys C重组蛋白对重组Cys C免疫小鼠血清的免疫反应性。结果 成功构建了重组表达载体pET30a-CysC,诱导表达出约19 ku的重组蛋白,经复性纯化后获得纯度达90%以上的目的蛋白,与重组Cys C免疫小鼠血清具有良好的免疫反应性。结论 在原核系统成功表达了人Cys C重组蛋白,而且具有良好的免疫反应性。更多还原

【Abstract】 Objective To obtain human cystatin C recombinant protein with immunoreactivity.Methods Total RNA was extracted from Hela cell and the human cystatin C gene fragment was obtained by RT-PCR and ligated into expression vector pET30 a.The recombinant pET30a-Cys C was transformed to E.coli BL21(DE3) and induced by IPTG.The expression product was renaturated by gel chromatography and purified by affinity chromatography.The immunoreactivity of recombinant Cys C was identified by ELISA against serum from the mouse immunized with recombinant Cys C.Results The recombinant expression vector pET30a-Cys C was successfully constructed and a 19 ku recombinant protein was induced.After renaturation and purification,recombinant protein with more than 90%purity was obtained and showed good immunoreactivity to serum from the mouse immunized with recombinant human Cys C.Conclusion The human Cys C recombinant protein was successfully expressed in prokaryotic expression system and showed good immunoreactivity.更多还原