人sTNFR1基因原核表达及体外抗肝细胞凋亡的活性研究

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L流式细胞术检测目的蛋白对TNF-α诱导QSG7701凋亡有抑制作用,这为今后的研究打下了基础.

Prokaryotic Expression of Human sTNFR1 Gene and the Anti-apoptosis Activity of its Recombinant Protein

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 identical 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 bound to sTNFR1's antibody in Western-blotting.Annexin V-FITC staining and flow-cytometry 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.