酵母双杂交法对HBV表面抗原主蛋白候选结合蛋白的筛选  

Screening of hepatocyte proteins interacting with the small surface protein of hepatitis B virus using yeast-two hybrid technique

作　　者：周飞[1,2,3,4] 任建林[1,2,3,4] 卢雅丕[1,2,3,4] 陈美娅 陈建民[1,2,3,4] 刘明[1,2,3,4] 施华秀[1,2,3,4] 张波[1,2,3,4] 董菁[1,2,3,4] 

机构地区：[1]厦门大学附属中山医院消化内科 [2]厦门大学消化疾病研究所 [3]厦门市消化疾病中心 [4]福建医科大学教学医院(厦门大学附属中山医院),福建省厦门市361004

出　　处：《世界华人消化杂志》2008年第13期1378-1382,共5页World Chinese Journal of Digestology

基　　金：厦门市首批重大疾病科研攻关资助项目;No.WKZ0501;厦门市卫生局医学科研立项资助项目;No.WSK0506;厦门大学引进人才科研启动基金资助项目;No.Z03109;福建省青年科技人才创新项目福建省高校新世纪人才创新资助项目~~

摘　　要：目的:自肝细胞cDNA文库中筛选与HBV主蛋白(SHBs)相互作用的蛋白基因,探讨主蛋白的生物学功能.方法:利用PCR扩增S主蛋白基因,定向克隆技术将靶基因克隆到pDEST32构建出S主蛋白的诱饵质粒;Westernblot方法验证转化诱饵质粒的酵母细胞表达SHBs;将诱饵质粒与人肝cDNA文库猎物质粒共同转化MaV203酵母细胞,在营养缺陷型培养基和X-gal上进行三重筛选阳性菌落,提取阳性酵母菌落的猎物质粒进行DNA测序;利用核苷酸数据库及生物信息学技术,对于筛选结果进行分析.结果:构建S主蛋白及肝文库酵母细胞表达载体,进行酵母双杂交系统筛选人肝细胞cDNA文库,筛选出既能在缺陷培养基也能在X-gal的检测下变成蓝色的真阳性菌落3个,分别为核糖体蛋白L3、微管蛋白α-1a和α-2巨球蛋白.结论:用酵母双杂交技术筛选出3个与SHBs相互作用的肝细胞结合蛋白编码基因,提示SHBs可能参与到肝细胞内部的多种生物学反应.AIM： To screen candidate hepatocyte binding proteins interacting with the surface antigenprotein （SHBs） of hepatitis B virus. METHODS： Polymerase chain reaction （PCR） was used to amplify SHBs gene. The target gene of SHBs was cloned into the yeast expression plasmid pDEST32 to construct bait plasmid pDEST32-SHBs. Western blot was employed to test SHBs expression after pDEST32-SHBs was transformed into the yeast cell MaV203 by Liacmediated method. Both pDEST32-SHBs and pDEST22-cDNA were contemporarily transformed into MaV203 cells to screen the binding protein of SHBs. MaV203 cells were plated on synthetic dropout nutrient media （SC/-Trp-Leu-His-Ura） and X-gal containing media for selection and screening. After that, the prey plasmids from true positive colonies were extracted and sequenced. The partial cDNA sequences in prey plasmids were analyzed by bioinformatics software. RESULTS： The yeast expression vector pD- EST32-SHBs was successfully constructed. After screening, 3 pieces of cDNA in prey plasmids from true positive blue colonies were sequenced. The cDNA sequences were alpha-2-macroglobulin, tubulin alpha 1a and ribosomal protein L3. CONCLUSION： Yeast-two hybrid method is successfully used for screening out alpha-2-macroglobulin, tubulin alpha 1a and ribosomal protein L3 as candidate binding proteins of SHBs.

关 键 词：乙型肝炎病毒 酵母双杂交技术 主蛋白 结合蛋白 聚合酶链式反应 

分 类 号：R373[医药卫生—病原生物学]