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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 ,克隆入原核表达载体pGEX_2T ,转化E .coliDH 5α ,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BGC82 3胃腺癌、A5 4 9肺腺癌、H6 9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
OBJECTIVE: To study the expression of mutant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and induce the apoptosis of various malignant cells in vitro so as to provide an experimental basis for the clinical treatment of malignant tumors by TRAIL. METHODS: The TRAIL oligonucleotides were synthesized in segments and 90 bases were changed. Then the full-length splicing was performed and cloned into the prokaryotic expression vector pGEX_2T. The recombinant plasmid was transformed into E. coli DH5α and induced with propyl thio-β-D-galactoside. Affinity chromatography separation and purification of fusion protein, thrombin enzyme digested TRAIL protein. MTT assay and flow cytometry quantitative analysis of purified products on a variety of tumor cells induced apoptosis. Results: The product of mutant TRAIL was soluble, and the inhibitory effect on human BGC82 3 gastric adenocarcinoma, A549 lung adenocarcinoma, H6 9 small cell lung cancer, KB nasopharyngeal carcinoma, A172 human glioma, SKNSH human neuroblastoma There is obvious induction of apoptosis. CONCLUSION: The prokaryotic expression mutant TRAIL has significant effect on inducing apoptosis of various malignant tumor cells