人新型肿瘤坏死因子在E.coli中的高效表达、纯化及诱导凋亡活性

人新型肿瘤坏死因子在E.coli中的高效表达、纯化及诱导凋亡活性 Expression of a Human TNF-α Mutant in E.coli and Aeasurement of Its Ability to Induce Apoptosis 王晓玲 WANG Xiao-ling 1 first-author 叶林柏 YE Lin-bai 1 郜金荣 GAO Jin-rong 1 徐进平 XU Jin-ping 1 曾晖 ZENG Hui 1 武汉大学病毒研究所,湖北,武汉,430072 武汉大学病毒研究所,湖北,武汉,430072 Institute of Virology, Wuhan University, Wuhan 430072, Hubie Institute of Virology, Wuhan University, Wuhan 430072, Hubie 采用 PCR技术获得了新型的人肿瘤坏死因子基因 ,在 15 3位氨基酸处引入突变 ,使 Gly突变为 L eu.将突变体 h TNF基因克隆到表达载体 p QE30中 ,SDS- PAGE结果显示经 IPTG诱导后 ,h TNF基因获得大量表达 .通过 Ni金属鳌合层析柱亲和层析获得到纯化的 h TNF融合蛋白 ,Western blot结果表明 h TNF蛋白可与抗TNF的抗血清发生特异性反应 .荧光染色实验检测了 h TNF蛋白诱导细胞发生凋亡的活性 Using PCR technique, a Tumor Necrosis Factor-α（TNF-α） mutant was obtained by altering the Gly on 153 site to Leu. After the amplified hTNF gene fragment was cloned into expressing vector pQE30, the E.coli transformed by the recombinant plasmid was induced by IPTG. SDS-PAGE assay proved that hTNF-αprotein expressed in E.coli reached a high level production. The protein was collected and highly purified through Ni-NTA-agarose. Westernblot showed that the protein could react strongly with specific anti-TNF serum. The ability of hTNF protein to induce apoptosis was examined using fluorescence staining method. 肿瘤坏死因子-α 表达 基因突变 凋亡活性 TNF-α expression gene mutagenesis apoptosis Q78 2001-02-01 2021-04-01 2