人sTNFR1基因的克隆、融合表达与生物学活性

Cloning , Expression of Human sTNFR1 Gene and the Biological Activity of Its Recombinant Protein

采用RT-PCR,从Hela细胞的mRNA中扩增人sTNFR1基因,构建含有目的片段的T载体克隆及原核表达载体pMAL-c2x重组质粒亚克隆,转化入大肠杆菌,测序证实其序列与基因数据库中sTNFR1基因一致。经异丙基-β-D半乳糖苷酶(IPTG)诱导表达,淀粉树脂亲和层析法纯化,得到融合蛋白sTNFR1-MBP。结果显示:经Western-blotting检测,sTNFR1-MBP具有免疫活性;MTT检测目的蛋白可有效地封闭TNFα对QSG7701的细胞毒性作用;流式细胞术检测目的蛋白对TNF-α诱导QSG7701凋亡有抑制作用;sTNFR1-MBP具有良好的生物活性,为今后的研究打下了基础。

Human sTNFR1 （soluble tumor necrosis factor receptor 1 ） gene was amplified by RT-PCR from Hela cells. A recombinant expression vector of sTNFR1-MBP was constructed in pMAL-c2x, and transformed into E. Coli JM109. It was sequenced and confirmed to be identifical to the sTNFR1 gene in data bank. Recombinant protein sTNFR1-MBP was induced by IPTG and purified by Amylose resin Affinity Chromatography. sTNFR1- MBP was binded to sTNFR1＇s antibody in Western-blotting. From MTT assays, the results showed that sTNFR1- MBP could effectively block the cytotoxicity mediated by TNFαon QSG7701 cells. Annexin V-FITC staining and flowcytometry were used to observe the recombinant protein＇s anti-apoptosis capacity and the recombinant protein has marked anti-apoptosis effect in vitro, sTNFR1-MBP had good biological activity and it will be employed in further study.