猪外周血单个核细胞cDNA酵母表达文库的构建及与猪瘟病毒E2蛋白相互作用细胞蛋白的筛选  

Construction of a yeast expression library of PBMC and screen of classical swine fever virus E2-interacting proteins

作　　者：廖亚金[1] 李素[1] 贺番[1] 何文瑞[1] 董泓[1] 冯烁[1] 孙元[1] 仇华吉[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2013年第9期707-710,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：中国农业科学院基本科研业务费预算增量项目(2013ZL034)

摘　　要：为研究猪瘟病毒(CSFV)蛋白与宿主细胞蛋白之间的相互作用,本研究通过SMART技术合成猪外周血单个核细胞(PBMC)双链cDNA,以携带与载体pGADT7-Rec重组位点同源序列的特异引物经long-distance PCR扩增双链cDNA。纯化双链cDNA并与载体pGADT7-Rec共转化酵母菌株Y187,经同源重组构建PBMC cDNA酵母表达文库。以CSFV E2蛋白为诱饵进行酵母双杂交筛选,得到阳性克隆根据序列比对分析结果,进一步进行共转化验证。结果显示筛选到17个与CSFV E2蛋白相互作用的宿主细胞蛋白,基因注释(GO)分析表明这些蛋白分别参与免疫、代谢、细胞生长与增殖、生物调节等过程,为研究它们与CSFV E2的相互作用奠定了基础。To investigate the interaction between classical swine fever virus （CSFV） proteins and host cellular proteins, the SMART technology, long-distance PCR and homology recombination technology were used to construct a porcine peripheral blood mononuclear cell （PBMC） cDNA library based on yeast strain Y187. In addition, a total of 17 E2-interacting proteins were screened from the library by yeast two-hybrid assay （Y2H） with CSFV E2 as bait. By using gene ontology （GO） analysis, 5 proteins was found to be involved in immune system process, 12 proteins were related in biological regulations. However, the exact functions of those proteins on CSFV replication require further investigations.

关 键 词：猪瘟病毒 PBMC CDNA文库 酵母双杂交 E2蛋白 

分 类 号：S852.65[农业科学—基础兽医学]