分泌型重组TRAIL基因真核表达载体的构建及其凋亡效应

Construction of the secretor type recombinant TRAIL gene eukaryotic expression vector and it′s effect on tumor cells

目的研究新型凋亡因子—TRAIL基因在肿瘤基因治疗中的作用。方法用RT-PCR法,从Balb/c小鼠脾脏的总RNA中扩增出编码凋亡诱导功能区的TRAILcDNA片段,并克隆入pUC-T载体中。经DNA测序鉴定正确后,亚克隆入真核表达载体pSEC-hy-gromycin的免疫球蛋白κ链信号肽编码区的下游,形成含有κ链信号肽与TRAIL凋亡诱导功能区的融合基因,构建可分泌表达TRAIL的重组真核表达载体pSEC/TRAIL。该重组载体经阳离子脂质体转染入小鼠前胃癌细胞株MFC615中,以台盼蓝拒染法和Hoechst33258荧光染色法,观察重组TRAIL对肿瘤细胞的凋亡效应。结果TRAIL的cDNA克隆成功,构建了可高效分泌表达可溶性TRAIL的重组真核表达载体pSEC/TRAIL,该产物能显著促进MFC615肿瘤细胞凋亡。结论重组真核表达载体pSEC/TRAIL的成功构建,为研究肿瘤的基因治疗提供了理想的工具。

Aim To investigate the role of the new apoptosis factor—TRAIL gene in tumor gene therapy. Methods The TRAIL cDNA fragment encoding apoptosis -inducing domain was amplified from the total RNA of Balb/c mouse′s spleen by means of RT-PCR. This RT-PCR product was cloned into sequencing vector pUC-T. After verified by sequencing, it was subcloned into the eukaryotic vector pSEC-hygromycin to form a eukaryotic vector pSEC/TRAIL secretorly expressing recombinant TRAIL gene. Results We had successfully cloned the TRAIL cDNA and constructed the highly secreted expressing TRAIL eukaryotic vector. This secreted recombinant TRAIL significantly induced the apoptosis of tumor cell MFC615 trasfected by its vector. Conclusion This paper provided excellent highly secreted expressing TRAIL eukaryotic vector for the research of anti-tumor gene therapy by TRAIL.