TRAIL 慢病毒载体构建及其在肝癌细胞中的表达

目的:构建携带TRAIL基因的慢病毒表达载体并实现其在肝癌细胞株HepG2中的稳定高表达。方法:构建TRAIL重组慢病毒表达载体pCDH-CMV-TRAIL-EF1-GFP-T2A-Puro,脂质体法将重组慢病毒载体和包装质粒混合物共转染293T细胞,包装产生慢病毒颗粒,纯化并测定病毒滴度。利用Western blotting检测TRAIL蛋白在HepG2中的表达。结果:酶切以及测序证实,成功构建TRAIL基因重组慢病毒载体,纯化的慢病毒滴度为1.02×104ifμ/μL。利用嘌呤霉素筛选获得稳定表达TRAIL的细胞系,经Western blot方法检测到TRAIL蛋白的稳定高表达。结论:成功构建了带有TRAIL基因的慢病毒载体,并实现其在HepG2的稳定高表达。

Construction of Lentiviral Vector Carrying TRAIL Gene and Its Expression in Hepatocarcinoma Cells

Objective:To construct lentiviral expression vector carrying TRAIL gene and realize its stable high expression in hepatocellular carcinoma cell line HepG2.Methods: PCDH-CMV-TRAIL-EF1-GFP-T2A-Puro recombinant lentiviral vector was constructed.The 293T cells were contransfected with the recombinant lentiviral vector and lentivirus package plasmid to produce lentiviral particles by lipofectamine method.The lentivirus was purified and virus titer was measured.Western blot was used to detect the expressions of TRAIL protein.Results: The recombinant lentiviral vector carrying TRAIL was confirmed by restriction end nuclease analysis and DNA sequencing.Virus titer reached to 1.02×10 4 ifμ/μL.After puromycin selection,the stable high expressions of TRAIL protein were confirmed by Western blotting.Conclusion: Lentiviral vector carrying TRAIL gene has been successfully constructed realizes its stable high expression in HepG2 cells.