人突变型TRAIL在大肠杆菌中的表达及诱导多种肿瘤细胞的凋亡

目的：研究突变型肿瘤坏死因子相关的凋亡诱导配体（TRAIL）的表达及体外诱导多种恶性肿瘤细胞的凋亡作用，为TRAIL临床治疗恶性肿瘤提供实验依据。方法：分段合成TRAIL寡核苷酸，并改变其中90个碱基，然后全长拼接，克服入原核表达载体pGEX-2T，转化E.coli DH 5a,丙基硫代－β-D－半乳糖苷诱导表达，亲和层析分离纯化融合蛋白，凝血酶酶切得到TRAIL蛋白。MTT法和流式细胞仪定量分析纯化产物对多种肿瘤细胞的诱导凋亡作用。结果：突变型TRAIL表达产物为可溶性，对人BGC823胃腺癌，A549肺腺癌，H69小细胞肺癌，KB鼻咽癌，A172人脑胶质细胞瘤，SKNSH人成神经细胞瘤株有明显的诱导凋亡作用，结论：原核表达突变型TRAIL具有明显的诱导多种恶性肿瘤细胞凋亡的作用。

Expression in Escherichia coli and apoptosis-inducing effect of recombinant TRAIL mutant on various malignant tumor cell lines in vitro

Objective:To study the apoptosis_inducing effects of human TNF_related apoptosis_inducing ligand(TRAIL) mutant expressed in Escherichia coli on various malignant tumor cell lines.Methods:The full_length of human TRAIL oligonucleotides was synthesized in segments with ninety of 843 bases being replaced to facilitate protein expression, and then the segments were linked together. Finally, the mutant TRAIL was inserted into the expression vector pGEX_2T and transformed into E.coli . The human TRAIL mutant proteins were expressed in E.coli induced by IPTG, and purified by affinity chromatography through GSTrap column. The simplex TRAIL mutant proteins were obtained by thrombin cleavage. The killing effect of TRAIL on the tumor cells was assessed by MTT, and apoptosis_inducing effect was quantified by flow cytometry analysis.Results:The TRAIL mutant proteins were soluble in water. Induction of apoptosis by the purified products was showed by a marked increase in apoptosis in human gastric adenocarcinoma cell line BGC823, pulmonary adenocarcinoma cell line A549, small cell lung cancer cell line H69, nasopharyngeal cancer cell line KB, malignant glioma cell line A172, and neuroblastoma cell line SKNSH, as compared with the controls.Conclusion:The human TRAIL mutant proteins have an obvious activity to induce apoptosis in various malignant tumor cell lines.