HCV包膜糖蛋白E2基因的克隆、蛋白表达及纯化

Cloning of HCV E2 gene and expression and purification of HCV envelope glycoprotein E2

目的:获得大量重组HCVE2蛋白,为研究E2蛋白的功能及制备其抗体奠定基础.方法:利用PCR方法从HCV基因组序列中扩增出831bp(384-661aa)的E2基因片段并按读框克隆到原核表达载体pET32a(+)上,得到重组质粒pET32a-HCVE2,转化大肠杆菌BL-21(DE3)菌株,IFFG诱导HCVE2蛋白表达,SDS-PAGE和Westernblot检测蛋白表达,Ni-NTA偶联的琼脂糖黏附柱纯化融合蛋白.结果:经IPTG诱导后,可见分子量约55000的融合蛋白表达;表达的蛋白主要以包涵体形式存在;经Ni-NTA偶联的琼脂糖黏附柱纯化的融合蛋白与抗His抗体及HCV阳性血清具有良好的反应原性.结论:HCVE2基因的克隆、表达及其融合蛋白的纯化为进一步开展HCVE2蛋白功能和HCV受体的研究奠定了基础.

AIM: To obtain large amount of HCV E2 protein, and to understand the function of the protein and to prepare the antibody against this protein. METHODS: A 831bp of E2 gene fragment was amplified by PCR method from HCV genome and cloned into pET32a (+) vector, an E.coli expression vector, to construct a re-combinant plasmid pET32a-HCVE2.The plasmid was transformed into Ecoli BL-21 (DE3) to express E2 protein with IPTG induced. The protein E2 fused with HIS tag was purification by Ni-NTA resin column. The protein E2 fused with His tag was detected by SDS-PAGE electrophoresis and Western blot. RESULTS: A novel protein with molecular weight of Mr 55 000 was expressed upon induction with IPTG in E.coli. The expressed product showed good reactivity to anti-His tag antibody and the HCV positive serum. CONCLUSION: Cloning of HCV E2 gene and the expression and purification of envelope glycoprotein E2 lay a foundation of further study on HCV E2 protein and the receptors of hepatitis virus C.