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

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4 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pCMV-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±2.5）%，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。

Abstract:

Objective To construct an eukaryotic expression vector of Hepatitis C virus(HCV) nonstructural protein NS4B, and investigate the effect of this protein on apoptosis of U2OS cells. Methods The fragment of target gene NS4B was obtained by PCR, and connected with the PCMV-tag2b vector using the homologous recombination method, and the PCMV-tag2b-NS4B was transformed into the DH5a. The right recombinant plasmid was selected. Lipofectamine 2000 was used to transfect PCMV-tag2b-NS4B into U2OS cells. Western blot was applied to detect the expression of HCV NS4B protein in U2OS cells, and immunofluorescence (IF) was used to assess the apoptosis of U2OS cells. Results The data showed that the recombinant plasmid PCMV-tag2b-NS4B was consistent with the one published on NCBI and could express HCV NS4B protein in U2OS cells. DAPI staining showed that the apoptosis rate of the PCMV-tag2b-NS4B group was (17.25 ±2.95)% and that of the control group was (6.53 ±2.36) %. Conclusions The eukaryotic expression vector of HCV NS4B has been successfully constructed, and HCV NS4B protein is expressed in U2OS cells and induces apoptosis.