欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (9): 180-184.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

重组人肿瘤坏死因子TNF-α的克隆与原核表达

李娜 李小刚 董岩岩 张程 孙玉科 刘钦松   

  • 收稿日期:2011-10-08 修回日期:2011-11-08 出版日期:2012-03-25 发布日期:2012-03-25

Cloning and Prokaryotic Expression of Recombinant TNF-α Gene

  • Received:2011-10-08 Revised:2011-11-08 Online:2012-03-25 Published:2012-03-25

PDF (PC)

1084

摘要：

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。

关键词: 耕作方式, 耕作方式, 冬小麦, 土壤含水量, 土壤温度, 容重, 杂草

Abstract:

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 can be purified and used to study its biological function, mechanism and prepare monoclonal antibody anti- TNF-α.