重组人肿瘤坏死因子TNF-α的克隆与原核表达

为了构建人肿瘤坏死因子TNF-α与谷胱甘肽-S-转移酶(GST)的融合基因表达载体，并进行原核表达。以人肿瘤坏死因子TNF-α全长转录本为模板，设计特异性引物，经PCR扩增目的基因将其定向克隆到pMD-l8T simple vector中，构建重组质粒pMD-18T-TNFα。然后通过亚克隆将测序正确的TNF-α基因插入表达载体pGEX-4T-1中，筛选出阳性质粒转化宿主菌株BL21(DE3)，通过温度、时间等不同条件诱导表达重组蛋白。结果表明，成功克隆到大小为702 bp的TNF-α全长cDNA序列，通过限制性酶切、PCR扩增证实pMD-18T-TNFα和pGEX-4T-1-TNFα载体已构建成功。诱导表达得到约51 kDa重组蛋白GST-TNFα。说明重组蛋白GST-TNFα的表达成功，为进一步获得纯化TNF-α，研究其生物功能、作用机理，制备抗TNF-α单克隆抗体奠定基础。

Cloning and Prokaryotic Expression of Recombinant TNF-α Gene

In order to construct human tumor necrosis factor TNF-α and glutathione-S-transferase（GST） fusion gene vector for prokaryotic expression.The open reading frame（ORF） of Human tumor necrosis factor TNF-α（TNF-α） gene was amplified by using specific primers and ligated to pMD-18T simple vector to generate pMD-18T-TNFα.Then it was released from the T simple plasmid by enzyme digestion and subsequently ligated to pGEX-4T-1,which had been digested with the same restriction enzyme,to create pGEX-4T-1-TNFα.To express GST-TNF α fusion protein in E.coli,pGEX-4T-1-TNFα was transformed to E.coli strain BL21（DE3） and the expression condition was optimized by adjusting tempreture,time and so on.SDS-PAGE result indicated that the 51 kDa fusion protein was expressed.It could be purified and used to study its biological function,mechanism and prepare monoclonal antibody anti-TNF-α.