酵母双杂交筛选与牛病毒性腹泻病毒NS4B互作的宿主蛋白  

Screening of host proteins interacting with bovine viral diarrhea virus NS4B by the yeast two hybrid system

作　　者：丁媛 石金凤 韩子琪 刁乃超 邓贺文 李健明[2,3,4] 时坤 杜锐[2,3,4,5] DING Yuan;SHI Jinfeng;HAN Ziqi;DIAO Naichao;DENG Hewen;LI Jianming;SHIKun;DU Rui(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;College of Chinese Medicine Materials,Jilin Agricultural University,Changchun 130118,China;Research Lab on Key Technology of Sika Deer Development and Resource Utilization,Changchun 130118,China;Jilin Province Sika Deer High-Efficiency Breeding and Product Development Technology Engineering Research Center,Changchun 130118,China;Key Laboratory of Animal Production and Product Quality and Safety,Ministry of Education,Changchun 130118,China)

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林农业大学中药材学院,吉林长春130118 [3]梅花鹿药用资源利用关键技术研究室,吉林长春130118 [4]吉林省梅花鹿高效养殖和产品开发技术工程研究中心,吉林长春130118 [5]动物生产及产品质量安全教育部重点实验室,吉林长春130118

出　　处：《中国兽医学报》2023年第2期269-277,共9页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31672577)。

摘　　要：为探究NS4B蛋白在牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)感染和复制过程中的作用,利用酵母双杂交(yeast two hybrid,Y2H)技术筛选与BVDV NS4B蛋白互作的宿主细胞蛋白质。以构建的BVDV NS4B重组质粒pGBKT7-NS4B作为诱饵质粒,采用酵母双杂交技术,与牛肾细胞MDBK-cDNA文库进行杂交。将获得的阳性克隆菌落经质粒抽提、测序分析和免疫共沉淀试验,确定可与NS4B互作的宿主细胞蛋白。结果表明,成功构建了诱饵质粒pGBKT7-NS4B,该质粒可在酵母菌中表达NS4B蛋白。采用酵母双杂交技术从牛肾细胞基因组文库获得14个阳性克隆,阳性克隆经测序、互补试验、免疫共沉淀验证后明确可与BVDV NS4B蛋白互作的宿主蛋白为SYNGR2和RABACI。上述研究为进一步研究BVDV NS4B蛋白在BVDV感染和复制中的作用机制奠定了理论基础。In order to explore the role of NS4B in the infection and replication of bovine viral diarrhea virus(BVDV),NS4B protein was regarded as the target,the host proteins which interacted with BVDV NS4B was screened with the help of the yeast two hybrid(Y2H)technique.The recombinant plasmid pGBKT7-NS4B was constructed as a bait to hybridize with the cDNA library of Madin-Darby bovine kidney(MDBK)by Y2H.After plasmid extraction,sequencing analysis and NCBI BLAST comparison of positive clones,the host proteins interacting with NS4B were finally identified.The results showed that the bait plasmid pGBKT7-NS4B expressing NS4B protein in Saccharomyces had been constructed successfully.There were 14 positive clones obtained from the screening in the genomic library of MDBK by Y2H.After the positive clones were verified by sequencing,complementation assay and immunoprecipitation,the host protein SYNGR2 and RABACI were identified to interact with BVDV NS4Bprotein.Above study laid a theoretical foundation for further understanding of the protein function of BVDV NS4B.

关 键 词：牛病毒性腹泻病毒 非结构蛋白4B 酵母双杂交 蛋白质互作 

分 类 号：S855.3[农业科学—临床兽医学]