人胱蛋白酶抑制剂C的原核表达及其免疫反应性研究  

Prokaryotic Expression and Immunoreactivity Investigation of Human Cystatin C

作　　者：杜惠芬[1] 付宝权[2] 李克生[1] 连晓雯[1] 王春芽 徐杨[1] 柴丹丹[1] 

机构地区：[1]甘肃省医学科学研究院,兰州730050 [2]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《现代检验医学杂志》2014年第1期32-34,37,共4页Journal of Modern Laboratory Medicine

基　　金：甘肃省科技支撑计划项目（No.1204FKCAl78）.

摘　　要：目的获得具有免疫反应性的人胱蛋白酶抑制剂C（Cysc）重组蛋白。方法从Hela细胞提取总RNA，采用RTPCR扩增获得人CysC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．coliBL21（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CysC重组蛋白对重组CysC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19ku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CysC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CysC重组蛋白，而且具有良好的免疫反应性。Objective To obtain human cystatin C recombinant protein with immunoreactivity. Methods Total RNA was ex- tracted from Hela cell and the human cystatin C gene fragment was obtained by RT-PCR and ligated into expression vector pET30a. 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 recombi- nant Cys C was identified by ELISA against serum from the mouse immunized with recombinant Cys C. Results The recom binant expression vector pET30a Cys C was successfully constructed and a 19 ku recombinant protein was induced. After re naturation 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.

关 键 词：人胱蛋白酶抑制剂C 原核表达 复性 免疫反应性 

分 类 号：Q784[生物学—分子生物学]