3种不同survivin基因启动子驱动Apoptin表达的重组慢病毒构建及体外抑瘤效果比较

Construction of three recombinant lentiviruses containing apoptin driven by survivin promoter of different lengths and comparison of their tumor suppressing efficency in vitro

目的构建3种不同长度的survivin基因启动子融合6×his标签Apoptin cDNA的重组慢病毒载体,经鉴定后行慢病毒包装并感染人结直肠癌细胞(SW480),体外实验观察并比较抑瘤效果。方法克隆3种不同长度的人survivin基因启动子(160、270、987 bp)和Apoptin-6×his cDNA,将其分别连接到pMD18-T载体并在该载体上完成survivin基因启动子和Apoptin基因组装。将组装好的表达盒构建入polyA改造后的慢病毒载体FG12中,利用三质粒包装系统包装成慢病毒。为观察重组慢病毒的抑瘤效果,以最适MOI值感染SW480肿瘤细胞后第5天及第30天,利用AnnexinV-PE/7-AAD凋亡试剂盒和流式细胞术检测细胞凋亡和周期变化,Western blot检测Apoptin-6×his表达。结果成功构建3种重组慢病毒载体,经包装浓缩后获得高滴度病毒,可以有效地感染目的细胞(感染效率大于80%)。体外结果提示病毒感染的SW480可以正确表达Apoptin-6×his蛋白,并能在感染后第30天出现明显的凋亡/坏死。其中,含有270 bp启动子的慢病毒抑瘤作用更为明显。结论采用270 bp survivin启动子,apoptin基因构建的重组慢病毒在靶细胞中显示出较好的抑瘤效果。

Objective To construct the recombinant lentivirus vectors using 3 survivin gene promoters （pSur） with a different length expressing 6 his-tagged apoptin cDNA and observe their tumor-suppressing effect after they were packed and human rectal cancer cells （ SW480 ） were infected with them. Methods Three survivin gene promoters at a length of 160, 270 and 987 bp expressing 6 his-tagged apoptin cDNA were cloned and linked to the pMD18-T vector on which survivin gene promoters and apoptin gene were assembled. The assembled expression box was constructed into the lentivirus vector FG12 modified with polyA. Recombinant lentiviruses were packed using the 3-plasmid packaging system. Tumor-suppressing effect of recombinant ]enti- virus vectors on SW480 cancer cells was observed while apoptosis and cell cycle of SW480 cancer cells were detected by Annexin V-PE/7-AAD and flow cytometry on days 5 and 30 after SW480 cancer cells were infected with recombinant lentiviruses at an optimal MOI. Results The 3 recombinant ]entivirus vectors were success- fully constructed, and high titer lentiviruses were obtained after they were packed and concentrated. The infec- tion rate of target cells for the purified lentivirus was over 80%. In vitro examination showed that the SW480 cancer cells expressed the apoptin protein with apoptosis or necrosis occurred on day 30 after they were infected with recombinant lentivirus. The tumor suppressing effect of recombinant lentivirus containing the 270 bp survivin promoter was more significant. Conclusion Recombinant lentivirus constructed with the 270 bpsurvivin promoter driving apoptin expression in target cells shows a rather good tumor-suppressing effect on human rectal cancer.