乙型肝炎病毒包膜大蛋白的自激活  

Autonomous activation of hepatitis B virus large surface protein

作　　者：纪冬[1] 成军[1] 陆荫英[1] 董菁[1] 郭江[1] 刘妍[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心全军病毒性肝炎防治研究重点实验室,北京市100039

出　　处：《世界华人消化杂志》2004年第10期2321-2324,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金攻关项目;No.C03011402;No.C30070689军队九五科技攻关项目;No 98D063军队回国留学人员启动基金项目;No.98H038军队十五科技攻关青年基金项目;No.01Q138军队十五科技攻关项目;No.01MB135~~

摘　　要：目的:构建乙型肝炎病毒包膜大蛋白的酵母表达载体,研究其自激活作用.方法:用多聚酶链反应(PCR)法扩增HBV包膜大蛋白(LHBs),前-S1蛋白(pre-S1),前-S2蛋白(pre-S2)及主蛋白(SHBs)的编码基因,并在其5’端引入EcoRI/BamHI内切酶位点,分别连接入酵母表达载体pGBKT7中,构建酵母表达质粒,转化酵母细胞AH109并在其内表达,然后分别铺于含有X-α-半乳糖的营养缺陷型培养基SD/-Trp和SD/-Trp-His-Ade上进行自激活验证(蓝/白筛选).结果:成功克隆出编码各种HBV包膜蛋白的基因,构建表达载体并在酵母细胞中表达,转化了pGBKT7-LHBs和pGBKT7-preS1的AH109酵母细胞在两种营养缺陷型培养基上均可正常生长,并且可以产生α-半乳糖苷酶从而在铺有X-α-半乳糖的培养基上呈现蓝色,而转化了pGBKT7-preS2和pGBKT7-SHBs的细胞只能在SD/-Trp的培养基上生长,且没有变蓝.结论:LHBs可以作为反式激活子发挥作用,代替GAL4蛋白的激活域来激活GAI4上游激活序列(GALUAS)和TATA盒,从而激活了下游报告基因(ADE2,HIS3,MEL1和LacZ)的表达,并且其自激活作用来自于前-S1结构域.AM: To construct the yeast expression vector of hepatitis B virus large surface protein (LHBs), and to study its autonomous activation. METHODS: The Matchmaker GAL4 two-hybrid technique was used. The LHBs, pre-S1, pre-S2 and SHBs genes were amplified by polymerase chain reaction (PCR) with respective primers. The amplified PCR fragments were then subcloned into the EcoR I/BamH I sites (5'ends) of pGBKT7 vector to obtain the expression vectors including pGBKT7 (-)-LHBs, pGBKT7(-)-preS1, pGBKT7(-)-preS2 and pGBKT7 (-)-SHBs. This vectors were identifed by PCR and digestion of EcoR I/BamH I. After the constructed vectors were transformed into yeast AH 109, the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing x-α-gal for testing their autonomous activation. RESULTS: The yeast expression vectors were constructed. The yeast cells transformed with pGBKT7-LHB and pGBKT7-preS1 vectors could grow well on both of the media. However, cells transformed with pGBKT7-preS2 and pGBKT7-SHBs vectors could only grow on the SD/-Trp medium. CONCLUSION: The LHBs functions as a transcriptional transactivator, and serves as the functional GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). The autonomous activation of LHBs roots in its pre-S1 domain.

关 键 词：乙型肝炎病毒 包膜大蛋白 自激活 前-S2蛋白 主蛋白 前-S1蛋白 

分 类 号：R373.21[医药卫生—病原生物学]