乙型肝炎病毒前S2基因酵母表达载体的构建及表达

Cloning and expression of PreS2 gene of hepatitis B virus in yeast

目的 探讨乙型肝炎病毒 (HBV)前S2蛋白的功能。方法 以质粒pCP10 (含有HBVayw亚型全长序列 )为模板 ,多聚酶链反应 (PCR)扩增HBV前S2基因 ,克隆到pGEM T载体中 ,测序鉴定、酶切后回收 ,与酵母表达质粒pGBKT7连接。将重组载体转化酵母细胞AH10 9,提取酵母蛋白质 ,进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western免疫印迹分析。结果 成功构建HBV前S2基因酵母表达载体 ,Western免疫印迹显示HBV前S2蛋白在酵母细胞中表达 ,表达产物在胞内存在 ,分子量 2 4kD左右。结论 HBV前S2蛋白在酵母细胞中表达成功。

Aim To investigate the gene expression of HBV PreS2 in yeast.Methods PCR was performed to amplify the gene of HBV PreS2 from the plasmid pCP10(containing the whole DNA fragment of HBV ayw subtype)and the PCR product was ligated into pGEM-T vector and identified by DNA sequencing.The gene of HBV PreS2 was cut from pGEM-T vector by enzyme digestion and cloned into yeast expression plasmid pGBKT7,then plasmid pGBKT7-PreS2 reconstructed was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Results HBV PreS2 gene was successfully cloned into pGBKT7 vector.The results of SDS-PAGE and Western blotting assay showed that HBV PreS2 protein was expressed in yeast cells and the molecular weight was about 23kD.Conclusion The findings suggest that HBV PreS2 is successfully expressed in yeast system.