【摘要】 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(114~281)和pET30a-TRAIL(120~281),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(114~281)(蛋白A),TRAIL(120~281)(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.更多还原

【Abstract】 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(114~281)and pET30a-TRAIL(120~281).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(114~281)\ and TRAIL(120~281)\ 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.更多还原