S100A11慢病毒表达载体的构建及其在肝癌细胞中的表达

目的 构建S100A11基因慢病毒表达载体,建立Huh-7 S100A11稳定细胞株,并鉴定其表达. 方法 RT-PCR扩增S100A11基因片段,与pWPT载体经Mlu Ⅰ和Not Ⅰ同时酶切后连接,构建慢病毒表达载体pWPT-S100A11;将表达载体pWPT-GFP和pWPT-S100A11与慢病毒包装质粒pMD2.G和psPAX2共同感染293T细胞,获得携带GFP和S100A11基因的慢病毒,将两种慢病毒分别感染Huh-7细胞,获得Huh-7 S100A11和Huh-7GFP稳定细胞株;利用Real-Time PCR和Western Blotting实验方法检测感染后S100A11的过表达情况. 结果 成功构建慢病毒表达载体pWPT-S100A11;慢病毒感染Huh-7细胞株后,阳性对照Huh-7GFP经荧光镜检测传染率达95％;感染Huh-7细胞后S100A11的mRNA和蛋白表达量均增加. 结论 成功构建慢病毒表达载体pWPT-S100A11,并建立了Huh-7S100A11稳定细胞株,为进一步研究S100A11基因在肝癌中的生物学功能和机制奠定了基础.

Construction of Lentivirus Vector S100A11 andits Expression in Hepatoma Cells

Objective To construct lentiviral vector carrying S100A11 gene and infected Huh-7 cells,and detect the expression of S100A11. Methods S100A11 gene was obtained by RT-PCR, and then cloned into the lentiviral vector pWPT digested by Mlu Ⅰ and NotⅠ to construct lentiviral vector pWPT-S100A11. The vector pWPT-GFP and pWPT- S100A11 with pMD2. G and psPAX2 co-infected 293T cells to get the recombinant lentivirus carrying GFP and S100A11 genes. The recombinant lentivirus carrying GFP and S100A11 were used to infect Huh-7 cells respectively. The expression of S100A11 in Huh-7 cells was detected by Real-Time PCR and Western blotting. Results The recombinant lentivirus ex- pression vector pWPT-S100A11 has been successfully constructed. The efficiency of recombinant lentivirus transfection reached to 95% under the green fluorescent. The mRNA and protein of S100A11 expression were all increased in Huh-7 cells after infection. Conclusion pWPT-S100A11 and Huh-7 S100A11 cells will be used in studying the function and mechanism of S100A11 in hepatic carcinomar.