HBeAg结合蛋白4剪切体HBeBP4A基因酵母表达载体的构建及表达研究  

Construction and expression of expressive vector of binding protein 4 spliced variant HBeBP4A of HBeAg in yeast cell

作　　者：张健康[1] 成军[2] 郭江[2] 刘春艳 赵龙凤[4] 洪源[2] 

机构地区：[1]山西医科大学第一医院消化内科,太原030001 [2]北京地坛医院传染病研究所 [3]空军太原干休所卫生所 [4]山西医科大学第一医院感染病科,太原030001

出　　处：《解放军医学杂志》2007年第3期213-215,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30371288);国家重点基础研究项目(973项目)(2004CB518908);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e抗原结合蛋白4基因剪切体HBeBP4A基因。方法以pcDNATM3.1/myc-HisA-HBeBP4A重组质粒作为模板,经RT-PCR扩增HBeBP4A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,行SDS-PAGE电泳和Western blotting分析。结果成功扩增出HBeBP4A基因,测序结果符合GenBank报告序列。酶切回收的HBeBP4A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 blotting分析显示该基因在酵母细胞中表达,表达产物相对分子量为61.37kD。结论成功构建了HBeBP4A酵母表达载体,并在酵母细胞中表达。Objective To study the exact function of HBeBP4A so as to investigate the gene expression of HBeBP4A in yeast cell. Methods Reverse transcription polymerase chain reaction （RT-PCR） was employed to amplify the gene of HBeBP4A from recombinant plasmids pcDNA 3. 1/myc-HisA-HBeBP4A, and the gene was cloned into pGEM-T vector. The gene of HBeBP4A was cut from pGEM- T-HBeBP4A vector and cloned into yeast expressive plasmid pGBKTT, and pGBKTT-HBeBP4A was then transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electmphoresis （SDS-PAGE） and Western blotting hybridization. Results HBeBP4A gene was successfully amplified and identified by DNA sequencing. The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109. The SDS-PAGE and Western blotting assay showed that the relative molecular weight of the expressed product was about 61.37kD, and HBeBP4A protein existed in yeast cells. Conclusion The findings suggested that HBeBP4A was successfully expressed into yeast system.

关 键 词：肝炎病毒 乙型 肝炎e抗原 乙型 剪切体 基因表达 

分 类 号：R341.3[医药卫生—基础医学]