应用酵母双杂交技术筛选人白细胞中与NS5ATP9蛋白结合蛋白的编码基因  

Screening and cloning of genes coding for leukocyte proteins interacting with NS5ATP9 by yeast-two hybrid technique

作　　者：李强[1] 梁耀东[1] 成军[1] 王琳[1] 张建[1] 邵清[1] 刘敏[1] 程明亮[2] 

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 [2]贵阳医学院第一附属医院感染科,贵州省贵阳市550004

出　　处：《世界华人消化杂志》2004年第4期828-831,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;No.C39970674;No.C39900130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"科技攻关面上项目;No.01MB135~~

摘　　要：目的:我们在以往的研究中,应用抑制性消减杂交技术(SSH)筛选得到了丙型肝炎病毒非结构蛋白5A反式激活蛋白9(NS5ATP9),NS5ATP9是一种未知功能新基因.为了进一步研究NS5ATP9的生物学功能,应用酵母双杂交技术, 筛选并克隆人白细胞中与NS5ATP9蛋白相互作用蛋白的基因,进一步阐明NS5ATP9的生物学功能及其作用途径. 方法:用多聚酶链反应(PCR)法扩增NS5ATP9基因,连接入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人白细胞cDNA文库质粒pACT2的酵母细胞Y187进行配合,在营养缺陷型培养基和X-α-半乳糖(X-α-gal)上进行双重筛选阳性菌落并测序,进行生物信息学分析. 结果:成功克隆出NS5ATP9基因并在酵母细胞中表达,配合后选出既能在四重缺陷(SD/-Trp-Leu-Ade-His)培养基上生长,又能在铺有C-α-gal的四缺培养基上变蓝的真阳性菌落46个,其中含人类免疫球蛋白轻链13个,核小体表面蛋白10个,铁蛋白重链2个,人类重组免疫球蛋白λ轻链11个,14-3-3家族蛋白1个,脑膜炎球菌PorA蛋白1 个,RNA多聚酶Ⅲ3个,烟草有丝分裂原激活蛋白激酶1 个,细胞色素P450Ⅱ2个,SLIT2蛋白1个,DNA依赖蛋白激酶催化亚基1个. 结论:成功克隆出丙型肝炎病毒NS5ATP9蛋白的结合蛋白,为进一步研究NS5ATP9的生物学作用提供了新的线索.AIM: To investigate the biological functions of NS5ATP9, and to screen proteins in leukocytes interacting NS5ATP9 protein by yeast-two hybrid. METHODS: The NS5ATP9 gene was amplified by poly-merase chain reaction (PCR) and NS5ATP9 bait plasmid was constructed by using yeast-two hybrid system 3, and the yeast AH 109 was then transformed. The transformed yeast mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu- His-Ade) containing X-a-gal for selecting two times and screening. After extracting and sequencing of plasmid DNA from blue colonies, we underwent analysis by bioinformatics. RESULTS: Forty six colonies were sequenced, among which thirteen colonies were Homo sapiens immunoglobulin light chain, ten ubiquitin, two ferritin heavy chain, eleven Homo sapiens rearranged immunoglobulin lambda light chain, one 14-3-3 family protein, one Meningococcus PorA protein, three RNA polymerase III, one tobacco mitogen activated protein kinase, two cytochrome P450 II, one SLIT2 protein, and one dependent-protein kinase catalylic subunit. CONCLUSION: Genes of NS5ATP9 interacting proteins in leukocytes are successfully cloned and the results bring some new clues for studying the biological functions of NS5ATP9 and associated proteins.

关 键 词：酵母双杂交技术 白细胞 NS5ATP9蛋白 抑制性消减杂交技术 SSH 编码基因 酵母细胞 

分 类 号：R346[医药卫生—基础医学]