重组可溶性人TRAIL抑制肿瘤细胞增殖

重组可溶性人TRAIL抑制肿瘤细胞增殖 Antitumor Activity of Recombinant Soluble Human TRAIL 王军 WANG Jun 1 2 first-author 于雯华 YU Wen-hua 1 2 康舒里 KANG Shu-li 1 2 肖庚富 XIAO Geng-fu 1 2 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 武汉大学生命科学学院,湖北,武汉,430072 武汉大学生命科学学院,湖北,武汉,430072 取健康人外周血得到总RNA,利用RT-PCR获取全长TRAILcDNA,以此为模板,在2对不同的引物下作PCR,得到不同长度的DNA片段,克隆到表达载体pET30a,得到重组质粒pET30a-TRAIL（114 2 81）和pET30a-TRAIL（120 2 81）,转化E.coliBL21（DE3）pLySs表达并纯化了2种不同长度的可溶性蛋白,分别是TRAIL（114 2 81）（蛋白A）,TRAIL（120 2 81）（蛋白B）.经MTT（四甲基偶氮唑盐）实验表明蛋白B能在6h内抑制不同类型的肿瘤细胞增殖,但其活性明显弱于蛋白A. As a potential antitumor protein, tumor necrosis factor-related apoptosis-inducing ligand（TRAIL） has drawn considerable attention. The coding region human soluble TRAIL gene from LPS activated healthy human peripheral lymphocytes （PBL） was cloned using RT-PCR and constructed 6 His-tag vector pET30a-TRAIL（114 2 81）and pET30a-TRAIL（120 2 81）.After DNA sequencing, The expression of protein was induced by 0.2 mmol/L IPTG and low temperature（4 ℃）. Soluble protein was purified by Ni-NTA chromatography column. Purified protein was digested with Enterkinase to cut off 6×His-tag and was purified by Gel filtration chromatography, showing a peak at 6.0×10 4 or so. Results of SDS-PAGE proved that the 6.0×10 4 protein was TRAIL trimer. The MTT assay showed that TRAIL（114 2 81）\ and TRAIL（120 2 81）\ could inhibit the growth of HeLa cell line, at the same time, protein B could inhibit the growth of some types of tumor cell and good relationship of concentration-effect and time\|effect. 肿瘤坏死因子相关的凋亡诱导配体 蛋白纯化 抗肿瘤活性 TRAIL protein purification antitumor activity R730.5 国家自然科学基金资助项目（30370054）;湖北省自然科学基金资助项目（2003S2146） 2005-04-01 2021-04-01 4