HCV Core基因真核表达载体构建及其对HeLa细胞自噬的影响  

Construction of eukaryotic expression vector of HCV Core gene and its influence on autophagy of HeLa cells

作　　者：王敏敏 魏建宏 任来峰 WANG Min-min;WEI Jian-hong;REN Lai-feng(Department of Nursing,Fenyang College Shanxi Medical University,Fenyang 032200,China;Department of Anatomy,Department of Basic Medicine,Fenyang College Shanxi Medical University;Immune Department,Shanxi Cancer Hospital)

机构地区：[1]山西医科大学汾阳学院护理系,032200 [2]山西医科大学汾阳学院基础医学部解剖教研室 [3]山西省肿瘤医院免疫室

出　　处：《天津医药》2019年第7期683-687,共5页Tianjin Medical Journal

基　　金：山西省自然科学基金项目(2014021037-9);山西省大学生创新创业训练计划项目(2014052)

摘　　要：目的构建丙型肝炎病毒(HCV)核心蛋白(Core)真核表达载体,并研究其对人宫颈癌HeLa细胞自噬功能的影响。方法以HCV复制子质粒pJFH1为模板,PCR法扩增HCVCore基因片段,经纯化回收后与PLVX-IRESZsGreen1载体用同源重组法相连,构建PLVX-IRES-ZsGreen1-Core重组质粒,并经测序验证其序列的正确性。转染宫颈癌HeLa细胞,并设PLVX-IRES-ZsGreen1-Core重组质粒转染组、PLVX-IRES-ZsGreen1空载体转染对照组以及未转染质粒空白对照组,通过免疫印迹法验证HCVCore蛋白在HeLa细胞中的表达,并用免疫荧光(IF)和免疫印迹检测自噬生物标记物微管相关蛋白1轻链3(LC3)的表达水平。结果获得PLVX-IRES-ZsGreen1-Core重组质粒,测序结果表明构建的重组质粒序列完全正确,HCVCore重组蛋白可在HeLa细胞中有效表达;免疫荧光和免疫印迹结果表明,HCVCore过表达细胞LC3焦点和LC3Ⅱ蛋白表达水平明显增多,PLVX-IRES-ZsGreen1-Core重组质粒转染组的细胞LC3Ⅱ水平(1.069±0.049)高于PLVX-IRES-ZsGreen1空载体转染对照组(0.776±0.047),差异有统计学意义(P<0.05)。结论成功构建了PLVX-IRES-ZsGreen1-Core重组质粒,HCVCore蛋白过表达可诱导HeLa细胞自噬。Objective To construct eukaryotic expression vector of hepatitis C virus (HCV) core protein, and investigate the effect of HCV Core protein on the autophagy of human cervical cancer HeLa cells. Methods The fragment of core was obtained by PCR from HCV replicon plasmid pJFH1. After purification and recovery, the DNA fragment was connected with the PLVX-IRES-ZsGreen1 vector using the homologous recombination method. DNA sequencing was used to verify the correctness of the sequence. PLVX-IRES-ZsGreen1-core was transfected into HeLa cells. Cells were divided into PLVX-IRES-ZsGreen1-core recombinant plasmid transfection group, PLVX-IRES-ZsGreen1 empty carrier transfection control group and control group without transfection plasmid blank. Western blot assay and immunofluorescence (IF) were applied to detect the expression of HCV Core protein and the autophagy-related biomarker (microtubule- associated protein 1 light chain3, LC3) in HeLa cells. Results PLVX-IRES-ZsGreen1-core recombinant plasmid was obtained. The sequencing results showed that the sequence of recombinant plasmid was completely corrected, and the HCV Core protein can be effectively expressed in HeLa cells. The results of Western blot assay and immunofluorescence also showed that LC3 Ⅱ protein level and LC3 foci were increased significantly in cells with HCV Core protein. The level of LC3 II was significantly higher in PLVX-IRES-ZsGreen1-Core recombinant plasmid transfection group (1.069±0.049) than that in PLVX-IRES-ZsGreen1 empty vector transfection control group (0.776±0.047, P<0.05). Conclusion The eukaryotic expression vector of HCV Core is successfully constructed, and the HCV Core protein can induce autophagy in HeLa cells.

关 键 词：丙型肝炎 肝炎病毒属 病毒核心蛋白质类 自噬 重组质粒 

分 类 号：R34[医药卫生—基础医学] R373.21