DLC-1基因重组慢病毒表达载体的构建及其在结直肠癌SW480细胞中的表达

Construction of recombinant lentiviral vectors expressing DLC-1 gene in colorectal carcinoma SW480 cells

目的:构建携带肝癌缺失基因-1(deleted in liver cancer-1,DLC-1)的重组慢病毒表达载体并实现DLC-1基因在结直肠癌(colorectal cancer,CRC)SW480细胞中的过表达。方法:将DLC-1基因的靶序列与载体质粒pcDNA3.1(+)-GFP连接成重组质粒。重组质粒经双酶切法及基因测序验证构建正确后包装成慢病毒颗粒并计算病毒滴度。重组慢病毒感染SW480细胞,同时设置阴性对照组和空白对照组流式细胞仪(flow cytometry,FCM)检测感染率;real-time PCR及Western blot检测SW480细胞中DLC-1基因表达水平。结果:重组质粒构建正确。重组慢病毒滴度为1×109 TU/ml;对SW480细胞的感染率为90%;DLC-1mRNA在重组慢病毒组细胞中过表达;重组慢病毒组DLC-1蛋白的表达水平显著高于阴性对照组(P=0.000);阴性对照组与空白对照组比较差异无统计学意义(P=0.902)。结论:成功构建了高滴度的DLC-1基因重组慢病毒,其携带的DLC-1基因能够在CRC SW480细胞中过表达,为后续研究DLC-1基因的表达在CRC的发生、发展中的作用奠定了实验基础。

Objective：To generate recombinant lentiviral vectors which can stably and effectively express DLC-1 gene in SW480 cells in colorectal cancer（CRC）. Methods：Target sequence of DLC-1 was connected with plasmid pcDNA3.1 （＋）-GFP to construct recombinant plasmid pcDNA3.1 （＋）-GFP-DLC- 1 and the integrity of pcDNA3.1 （＋）-GFP-DLC- 1 was detected. Recombinant lentiviral vectors containing the recombined plasmids were packaged and the virus titer was calculated. The SW480 cells were infected by the recombinant lentiviral vectors and the transfection efficiency was detected. Negative control and blank control were set at the same time 109. Then real-time PCR and Western blot were employed to evaluate expression level of DLC-1 gene by the recombinant lentiviral vectors in SW480 cells. Results：Recombinant lentiviral vectors were correctly constructed. The virus titer was 1×10^9 TU/ml and the transfection efficiency was 90%. Real-time PCR revealed overexpression of DLC-1 mRNA in the transfected SW480 cells. Western blot got the same result that the expression level of DLC-1 gene in the transfected SW480 cells was significantly higher than that of negative control group（P=-0.000） and difference between blank control group and negative control group was not significant（P=0.902）.Conclusions：Recombinant lentiviral vectors containing DLC-1 gene are successfully constructed and DLC-1 gene is stably and effectively overexpressed in the transfected SW480 ceils, which lay foundation for further research on roles of DLC-1 gene in the development and progression of CRC.