【摘要】 探讨构建人脂多糖诱导肿瘤坏死因子释放因子(LITAF)基因的过表达慢病毒载体的技术方法,并检测其体外表达目的基因的水平。设计LITAF基因引物,应用聚合酶链反应(PCR)的方法扩增LITAF基因片段;应用EcoRI、Bam HI酶切LV8载体,通过连接酶将LITAF基因片段连接至线性化的LV8载体上,应用酶切及测序方法鉴定LITAF-LV8重组质粒,将其包装慢病毒后感染293T细胞(人胚肾细胞),观察绿色荧光蛋白(GFP)的表达,验证后感染肝癌细胞株HepG2。RT-PCR和Westernblot鉴定感染后HepG2中LITAF的表达。LITAF基因在慢病毒感染的HepG2细胞中的表达显著高于对照组细胞。说明成功构建了过表达LITAF的HepG2细胞株,为后期研究提供了实验基础。更多还原

【Abstract】 To construct a Ientivirus-mediated overexpression vector containing LITAF,and examine its ability to express LITAF in vitro,the primers of LITAF gene were designed for amplification of LITAF gene fragment by polymerase chain reaction( PCR). The LV8 vectors were digested by EcoRI、Bam HI,which was then linked to the full length of LITAF gene fragements by ligase. Postive clones of LITAF-LV8 vectors were identified by DNA sequencing。According to the packaging kit manual,the lentiviral vectors containing LITAF gene were transfected into293 T cells for lentivirus package. Observation of the expression of GFP,then transfected into HepG2 cells. The recombinant plasmid was confired by PCR and Western blot. The lentivirus expression vector containing LITAF was successfully constructed which could be stably overexpressed in human liver cancer cell HepG2. It is conclused the lentiviral vector over-expression LITAF gene was successfully constructed,which provided an experimental foundation for further studies of LITAF.更多还原