HCV1b型NS4B基因慢病毒载体构建及其在LO_2细胞中的表达

Construction of lentiviral vectors containing HCV 1b type nonstructural protein 4B gene and its overexpression in LO_2 cells

目的构建HCV1b型非结构蛋白4B(NS4B)基因与红色荧光蛋白mkate2基因融合表达的慢病毒载体和慢病毒稳定细胞株LO2NS4B,为深入研究HCVNS4B的功能及其致病机制提供理想的细胞模型。方法以HCV1b NS4B全长克隆为模板扩增NS4B基因,并与荧光蛋白mkate2基因融合,通过限制性内切酶BamHⅠ、AscⅠ酶切和T4 DNA连接酶连接,将NS4B-mkate2融合基因插入慢病毒载体pLenti6.3-MCS,构建PL6.3-NS4B-mkate2重组质粒,经PCR及测序鉴定重组质粒正确后通过脂质体将其与包装质粒共转染293FT细胞,进行慢病毒包装并测定病毒滴度,以感染复数(MOI)为50感染LO2细胞,荧光显微镜下观察感染效率,经Blasticidin筛选,获得表达NS4B基因的慢病毒稳定细胞株LO2NS4B。经QPCR和Western blotting分别检测LO2NS4B中NS4B mRNA和NS4B-mkate2融合蛋白的表达情况。结果成功构建慢病毒表达载体PL6.3-NS4B-mkate2,与包装质粒共转染293FT细胞可见大量红色荧光,浓缩病毒后测定其滴度为1.815×108TU/mL;以MOI为50感染LO2细胞,经过Blasticidin筛选,感染效率在90%以上;QPCR和Western blotting分别证实LO2细胞表达NS4B mRNA和蛋白。结论成功构建表达NS4B的慢病毒稳定细胞株LO2NS4B,为深入研究HCVNS4B基因在HCV慢性化和肝癌发生、发展中的作用奠定了基础。

[Objective] To construct lentiviral vectors containing HCV lb type nonstructural protein 4B （NS4B）-mkate2 gene and LO2 stable cell lines overexpressing NS4B gene, for further investigation of its function and pathogenic mechanism provides an ideal cell model. [Methods] HCVlb NS4B gene was amplified from a plasmid containing the full-length NS4B sequence and fused with mkate2 gene and cloned into the lentiviral vector pLenti6.3-MCS by restriction endonuclease BamHI and AscI digestion and T4 DNA ligase ligation. After transformation into competent E.coli cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the packaging plasmids were co-transfected into 293F1＂ cells by lipo- feetamine 2000 to produce the lentiviral particles, and the viral titer was detected. The LO2 cells were infect- ed by the lentiviral particles obtained at a multiplicity of infection （MOI） of 50, and the transfection efficiency was assessed under fluorescent microscope. LO2 stable cell lines expressing NS4B gene were screened by blasticidin. QPCR and Western blotting were used to detect the expression of NS4B mRNA and protein in the transfeeted LO2 cells, respectively. [Results] The lentiviral vector PL6.3-NS4B-mkate2 for NS4B gene was successfully constructed. Strong red fluorescence was observed in 293PI＇ cells under fluorescent microscope af- ter eo-transfeetion of the cells with the recombinant plasmid and the packaging plasmids. The virus in the supernatant reached a titer of 1.815~108 TU/mL. The transfection efficiency of the collected virus exceeded 90% in LOs cells at a MOI of 50 by blasticidin selection. QPCR and Western blotting identified the mRNA and protein expression of NS4B in the transfected LO2 cells respectively. [ Conclusion] LO2 cell lines for NS4B gene stable expression have been successfully established, which facilitate further investigation of the roles of NS4B gene in the ehronicity of HCV infection and the development and progression of hepatoeellular carcinoma.