乙型肝炎病毒前S1蛋白反式激活基因5在酵母细胞中的表达  

Expression of human gene 5 transactivated by pre-S1 protein of hepatitis B virus in yeast cell

作　　者：张健康[1] 成军[2] 郭江[2] 伦永志[2] 王丹琼[2] 赵龙凤[3] 洪源[2] 毛羽[2] 

机构地区：[1]山西医科大学第一医院消化科,太原030001 [2]北京地坛医院传染病研究所 [3]山西医科大学第一医院感染病科,太原030001

出　　处：《中华传染病杂志》2007年第11期641-644,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金(30371288);北京市自然科学基金项目(5042024)

摘　　要：目的在真核生物酵母细胞中表达乙型肝炎病毒前S1蛋白反式激活基因5(PS1TP5)。方法以HepG2细胞来源的mRNA为模板,经RT-PCR法扩增PS1TP5基因,克隆到pGEM-T载体中,并测序鉴定,酶切回收后连接到酵母表达质粒pGBKT7中,并转化酵母AH109,色氨酸缺陷型培养基(SD/-Trp)上筛选阳性菌落,提取酵母蛋白质,进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western免疫印迹分析,以明确PS1TP5是否可在其中表达。结果成功扩增出PS1TP5,测序鉴定符合基因库报告序列,酶切回收的PS1TP5基因片段成功克隆入酵母表达载体pGBKT7,并转化入酵母细胞AH109中,Western免疫印迹显示该基因在酵母细胞中成功表达,表达产物相对分子质量为36950。结论成功构建了PS1TP5酵母表达载体,并在酵母细胞中表达。Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis E virus （PS1TP5） by investigating the gene expression of PS1TP5 in yeast ceils. Methods Reverse transcription-polymerase chain reaction（RT-PCR） was performed to amplify the gene of PSITP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector. The gene of PSITP5 was cut from pGEM-T-PSITP5 vector and cloned into yeast expressive plasmid pGBKT7, then pGBKT7-PS1TP5 was transformed into yeast cell AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western hybridization. Results PSITP5 gene was successfully amplified and identified by DNA sequencing. The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950, and PSITP5 protein existed in yeast cells. Conclusion The findings suggest that PSITP5 can be successfully expressed in yeast cell.

关 键 词：肝炎病毒 乙型 前S1蛋白 反式激活蛋白 酵母菌 基因表达 

分 类 号：R512.6[医药卫生—内科学] R346[医药卫生—临床医学]