HIV跨膜蛋白gp36和gp41的截短及融合表达

目的将HIV-1的跨膜蛋白gp41和HIV-2跨膜蛋白gp36进行截短,并在大肠杆菌中进行融合表达。方法用PCR将gp41和gp36的编码基因进行截短,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用BamHⅠ、EcoRⅠ和SalⅠ切下目的基因,并构建到表达载体pGEX-4T-3,导入宿主细胞BL21,用IPTG诱导表达。结果酶切鉴定显示,截短的HIV-1gp41和HIV-2gp36跨膜蛋白基因大小与预期的一致,表达产物经SDS-PAGE分析显示在相对分子质量66000处出现融合表达条带,Westernblot分析显示,与相应抗体出现特异性反应。结论已成功对gp41和gp36跨膜蛋白进行截短,并构建表达载体进行表达,为跨膜蛋白的进一步应用研究奠定基础。

Truncation and Fusion Expression of Tans-membrane Proteins gp36 and gp41 of HIV

Objective To truncate the trans-membrane proteins gp41 of HIV-1 and gp36 of HIV-2 and express in E.coli.Methods Truncate the genes encoding gp41 and gp36 by PCR,purify the PCR product and clone into vector pGEM-T.Digest the recombinant plasmid with BamHⅠ,EcoRⅠ and SalⅠ and insert the obtained target gene into expression vector pGEX-4T3.Transform the constructed recombinant plasmid to E.coli BL21 for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.Results Restriction map proved that the lengths of truncated gp41 and gp36 genes were identical to those expected.SDS-PAGE profile revealed a fusion expression band with a relative molecular weight of 66 000.Western blot showed specific reactions of expressed product with the corresponding antibodies.Conclusion The HIV-1 gp41 and HIV-2 gp36 genes were successfully truncated and expressed in E.coli.It laid a foundation of further application of trans-membrane protein.