利用酵母双杂交技术筛选与新孢子虫NcSRS2蛋白相互作用的宿主蛋白  

Screening of host proteins interacting with Neospora Caninum NcSRS2 protein by yeast two⁃hybrid system

作　　者：金春梅[1,2] 周博宇 姚远 吴保义 李红旺[1] 王楠[3] 于龙政 JIN Chunmei;ZHOU Boyu;YAO Yuan;WU Baoyi;LI Hongwang;WANG Nan;YU Longzheng(Agriculture College,Yanbian University,Yanji 133002,China;Engineering Research Center of Beef Cattle Science and Technology Innovation of Ministry of Education in Northeast Cold Region,Yanji 133002,China;Jilin Province Animal Husbandry and Veterinary Academy of Sciences,Changchun 130062,China)

机构地区：[1]延边大学农学院,吉林延吉133002 [2]东北寒区肉牛科技创新教育部工程研究中心,吉林延吉133002 [3]吉林省畜牧兽医科学研究院,吉林长春130062

出　　处：《甘肃农业大学学报》2023年第3期1-6,共6页Journal of Gansu Agricultural University

基　　金：国家自然科学基金项目(32060807);吉林省教育厅“十三五”科学技术项目(JJKH20200522KJ);高等学校学科创新引智计划(D20034)。

摘　　要：【目的】筛选与鉴定与新孢子虫NcSRS2蛋白相互作用的Vero细胞蛋白。【方法】从Nc1株犬新孢子虫速殖子中提取DNA作为模板,PCR扩增NcSRS2基因,克隆于pGBKT7表达载体,构建诱饵载体pGBKT7-NcSRS2,转化入Y2H酵母感受态细胞,进行自激活活性验证和细胞毒性检测。利用酵母双杂交技术,以转化诱饵载体pGBKT7-NcSRS2的Y2H酵母菌株与Vero细胞cDNA文库进行酵母双杂交,筛选与NcSRS2蛋白相互作用的宿主蛋白,对阳性克隆进行测序,并用BLAST分析,筛选出与NcSRS2存在相互作用的Vero细胞蛋白质。【结果】诱饵载体pGBKT7-NcSRS2无自激活活性和细胞毒性,可用于文库筛选。运用酵母双杂交系统,筛选出2个与NcSRS2相互作用的Vero细胞成分,分别是NADH dehydrogenase subunit 1和STMN2。【结论】本研究首次运用酵母双杂交技术筛选出与新孢子虫NcSRS2相互作用的Vero细胞蛋白质,为进一步研究犬新孢子虫NcSRS2参与黏附及入侵宿主细胞的机制奠定基础。【Objective】The study aim to screen and identify Vero cell proteins that can interact with Neospora caninum NcSRS2 protein.【Method】The DNA is extracted from the Nc1 strain of N.caninum as a template.The NcSRS2 gene is amplified by PCR,and cloned into the pGBKT7 expression vector to construct the yeast two-hybrid bait vector pGBKT7-NcSRS2.And then,the pGBKT7-NcSRS2 is transformed into the yeast competent cell Y2H.The self-transcriptional activity and cytotoxicity of the constructed bait vector pGBKT7-NcSRS2 are detected.The yeast cell of Y2H containing the pGBKT7-NcSRS2 bait vector is cultured with the Vero cell cDNA library to screen the host proteins interacting with NcSRS2 by yeast two-hybrid technology.The positive clones are sequenced for the BLAST analysis.【Result】The bait vector pGBKT7-NcSRS2 is no self-transcriptional activity and cytotoxicity and can be used for library screening.Two prey protein interacting with NcSRS2 are captured from Vero cell including NADH dehydrogenase subunit 1 and STMN2.【Conclusion】This study firstly report on the screening of Vero cell proteins interacting with Neospora caninum NcSRS2 protein by yeast two-hybrid technology,which provide a scientific basis for studying the mechanism of N.caninum adhesion and invasion of host cells.

关 键 词：犬新孢子虫 NcSRS2 酵母双杂交 筛选 

分 类 号：S852.723[农业科学—基础兽医学]