人胱抑素C的原核表达及包涵体复性研究

Analysis and Inclusion Bodies Refolding of Human Cystatin C Expression in Prokaryotic Cells

构建胱抑素C原核表达载体p Cold1-cysc,p ET28a-cysc,p ET32a-cysc和p ET41a-cysc及相应的BL21(DE3)表达菌株,最终得到重组人胱抑素C蛋白。应用基因工程手段,原核表达,亲和层析及透析复性的方法对目的蛋白进行表达,纯化和复性。结果:经分析后,选取表达量较大的菌株BL21(DE3)p ET32a-cysc和BL21(DE3)p Cold1-cysc,对目的蛋白进行提取及纯化,对纯化后的可溶及包涵体形式的重组胱抑素C进行透析复性及免疫活性的检测。Western blotting及ELISA结果显示,携带有不同融合标签的可溶性重组胱抑素C或包涵体在透析复性后具有不同的免疫活性。结论:在大肠杆菌中成功表达并得到人胱抑素C的蛋白,复性后得到了重组胱抑素C的活性形式。

Through construction of Cystatin C （without signal peptide） prokaryotic expression vectors pColdl-cysc, pET28a-cysc, pET32a-cysc and pET41a-cysc got the recombinant protein Cystatin C. The gene engineering,prokaryotic expression ,affinity chromatography and dialysis refolding were mainly used. Results showed that the strain BL21 （ DE3 ） pET32a-cysc and BL21 （ DE3 ） pCold1- cysc in which recombinant protein Cystatin C can express well were chosen to cuhure and the inclusion bodies of recombinant protein Cystatin C were isolated and purified by using IPTG induction and affinity chromatography respectively. The different express vectors have various effects on heterologous protein expression in E. coli BL21 （DE3） and the soluble or refolded inclusion bodies of recombinant Cystatin C containing different protein tags have different activities after refolding. The recombinant protein of human Cystatin C was expressed successfully in E. coli BL21 containing plasmid pET32a-cysc or pCold1-cysc, and the recombinant protein purified containing biological activity was got through purification and refolding.