人CD81细胞外区大环原核表达载体的构建及在大肠杆菌中的表达和纯化

目的:构建人CD81-LEL基因的原核表达载体,并在大肠杆菌中进行表达. 方法:通过PCR扩增得到CD81-LEL基因编码区片段,与pMD18-T载体连接,经序列测定后,再将该扩增片段亚克隆入原核表达载体pET32a( )中.酶切鉴定正确的表达载体pET32a-CD81-LEL转化表达菌BL21(DE3)进行诱导表达,用SDS-PAGE和Western-blot对表达产物进行鉴定,用镍离子亲和层析法纯化融合蛋白. 结果:成功构建了原核表达载体pET32a-CD81-LEL;SDS-PAGE显示目的蛋白大量表达且呈可溶性状态,Western-blot显示目的蛋白正确表达,并用镍离子亲和层析法获得纯化的重组蛋白. 结论:CD81-LEL可以在大肠杆菌中表达,为进一步研究CD81与丙型肝炎病毒(HCV)包膜糖蛋白E2的结合提供了有用的研究资料.

Cloning and expression of the human CD81-LEL gene in E. coli

Objective: To construct a recombinant prokaryotic plasmid of the CD81-LEL gene and observe its expression in E.coli.Methods: The CD81-LEL gene fragment was amplified by PCR,and subsequently cloned into the pMD-18T vector.After verification by sequencing,it was subcloned into the plasmid pET32a(+).The recombinant plasmid pET32a-CD81-LEL was then transformed into E.coil BL21(DE3) to express CD81-LEL induced with IPTG.The expression product was detected by SDS-PAGE and Western blot.The protein CD81-LEL fused w...