HCV NS4B表达载体的构建及其对细胞凋亡的影响  

Construction of expression vector of HCV NS4B and its effect on cell apoptosis

作　　者：宋彬妤[1] 赵嘉伟[2] 宋静 陈思思 胡志廷 韩昕 王敏敏[1] 任来峰 

机构地区：[1]山西医科大学汾阳学院科技中心,山西汾阳032200 [2]厦门大学医学院临床医学系,福建厦门361002 [3]山西医科大学汾阳学院医学检验系,山西汾阳032200

出　　处：《中国现代医学杂志》2017年第26期25-29,共5页China Journal of Modern Medicine

基　　金：山西省自然科学基金(No:2014021037-9);山西省高等学校大学生创新创业训练项目(No:2014052);山西医科大学汾阳学院科技发展基金(No:2016B02)

摘　　要：目的构建丙型肝炎病毒(HCV)2a型非结构蛋白4B(NS4B)的真核表达载体,并观察其对骨肉瘤细胞U2OS凋亡的影响。方法以质粒PJFH1为模板,通过PCR反应扩增NS4B目的基因片段,采用同源重组法与PCMV-tag2b载体相连,转化大肠杆菌感受态DH5α,筛选正确的克隆。以脂质体为介导转染U2OS细胞,通过Western blot检测NS4B蛋白的表达,免疫荧光检测细胞的凋亡。结果构建p CMV-tag2b-NS4B重组质粒,经测序其与NCBI公布的序列完全一致,并可在U2OS细胞中表达,DAPI检测显示细胞凋亡率为(17.25±2.95)%,对照组凋亡率为(6.53±2.36)%。结论成功构建NS4B的真核表达载体,并可在U2OS细胞中表达,诱导细胞凋亡。Objective To construct an eukaryotic expression vector of Hepatitis C virus(HCV)nonstructural protein NS4 B, and investigate the effect of this protein on apoptosis of U2 OS cells. Methods The fragment of target gene NS4 B was obtained by PCR, and connected with the PCMV-tag2 b vector using the homologous recombination method, and the PCMV-tag2 b-NS4 B was transformed into the E.coli DH5 a. The right recombinant plasmid was selected. Lipofectamine 2000 was used to transfect PCMV-tag2 b-NS4 B into U2 OS cells. Western blot was applied to detect the expression of HCV NS4 B protein in U2 OS cells, and immunofluorescence(IF) was used to assess the apoptosis of U2 OS cells. Results The data showed that the recombinant plasmid PCMV-tag2 b-NS4 B was consistent with the one published on NCBI and could express HCV NS4 B protein in U2 OS cells. DAPI staining showed that the apoptosis rate of the PCMV-tag2 b-NS4 B group was(17.25 ± 2.95) % and that of the control group was(6.53 ± 2.36) %. Conclusions The eukaryotic expression vector of HCV NS4 B has been successfully constructed, and HCV NS4 B protein is expressed in U2 OS cells and induces apoptosis.

关 键 词：丙型肝炎病毒 非结构蛋白4B 重组质粒 凋亡 

分 类 号：R373.21[医药卫生—病原生物学]