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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。
In order to express the human soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein in Escherichia coli, the cDNA of human soluble TRAIL protein was amplified from total RNA of activated human peripheral blood lymphocytes by RT PCR and cloned into the PCR2 1 vector After sequencing, the eukaryotic and prokaryotic expression plasmid vectors of human soluble TRAIL protein were constructed respectively by gene recombination method. The recombinant plasmids were transformed into COS 7 and E. coli M1 5 respectively. The transient expression of human soluble TRAIL protein in COS 7 cells was detected by flow cytometry. The expressed products of E. coli were identified by SDS PAGE electrophoresis and Western blot. The expressed fusion protein is a human soluble TRAIL protein molecule with a relative molecular mass of 210,000 and an expression level of about 2 mg / ml. The expressed human soluble TRAIL protein has the effect of inducing HL 60 cell apoptosis. The above results suggest that E. coli can express bioactive human soluble TRAIL protein, which provides a material for further study on the possible application of TRAIL in tumor and autoimmune diseases.