人可溶性TRAIL基因的克隆及表达

The Cloning and Expression of Human Soluble TRAIL in E.coli

为了利用大肠杆菌表达人可溶性肿瘤坏死因子相关性凋亡诱导配体 (TRAIL )蛋白 ,应用RT PCR技术从激活的人外周血淋巴细胞总RNA中扩增人可溶性TRAIL蛋白cDNA ,克隆入PCR2 1 载体 ,测序验证后用基因重组法分别构建了人可溶性TRAIL蛋白的真核与原核表达质粒载体。将重组质粒分别转入COS 7和大肠杆菌M1 5中表达。用流式细胞仪检测人可溶性TRAIL蛋白在COS 7细胞中的瞬时表达 ,用SDS PAGE电泳和Westernblot鉴定大肠杆菌中的表达产物。所表达融合蛋白为人可溶性TRAIL蛋白分子 ,相对分子质量为 2 1 0 0 0 ,表达量约为 2mg/ml。所表达的人可溶性TRAIL蛋白具有诱导HL 60细胞凋亡的作用。上述结果提示大肠杆菌可良好表达具有生物活性的人可溶性TRAIL蛋白 ,为深入研究TRAIL分子在肿瘤与自身免疫性疾病中的可能应用提供了材料。

To obtain recombinant human soluble TRAIL molecule,RT PCR was applied to amplify cDNA of soluble TRAIL molecule from the total RNA extracted from activated human peripheral blood lymphocytes The DNA fragment was cloned into PCR 2 1 vector After sequencing,the cDNA of soluble TRAIL molecule was inserted into pTarge and pQE vector and expressed in COS 7 and E coli M15 respectively The protein expressed on COS 7 was identified by FACS and by SDS PAGE and Western blotting after purified through affinity chromatography column with ligand of 6× His tag respectively The protein expressed was purified with one band which was 21 000 analyzed by SDS PAGE The expressing protein was soluble TRAIL molecule which identified by Western blot The molecule expressed could induce apoptosis of tumor cell lines The fusion human soluble TRAIL molecule was obtained It lays the foundation for the further studying