猪小肠上皮细胞酵母双杂交cDNA文库的构建  

CONSTRUCTION AND CHARACTERIZATION OF THE YEAST TWO-HYBRID CDNA LIBRARY OF SWINE SMALL INTESTINAL EPITHELIAL CELLS

作　　者：李慧春[1] 陈鹏飞[1] 李先斌[1] 丁思嘉[2] 王康[1] 张文超[1] 杨德强[1] 李林[1] 徐晶晶[1] 虞凌雪[1] 姜一峰[1] 高飞[1] 于海[1] 童光志[1,3] 周艳君 LI Hui-chun;CHEN Peng-fei;LI Xian-bin;DING Si-jia;WANG Kang;ZHANG Wen-chao;YANG De-qiang;LI Lin;XU Jing-jing;YU Ling-xue;JIANG Yi-feng;GAO Fei;YU Hai;TONG Guang-zhi;ZHOU Yan-jun(Shanghai Veterinary Research Institute, CAAS,Shanghai 200241,China;School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]上海交通大学农业与生物学院,上海200240 [3]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2019年第2期19-24,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金项目(31472207);国家重点研发计划项目(2016YFD0500100);国家生猪现代产业技术体系建设项目(CARS-36)

摘　　要：猪小肠上皮细胞(intestinal epithelial cell,IECs)是猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)在宿主体内感染的靶细胞,为了研究PEDV在体内与宿主细胞蛋白之间的互作关系以便于揭示其致病机理,本研究以IECs为研究对象,提取了IECs的总RNA,利用SMART技术经过反转录、扩增和纯化获得双链cDNAs后,将其与pGADT7-Rec和carrier DNA共同转化至酵母Y187菌株,进行选择性培养基(SD/-Leu)筛选以及文库容量及质量的鉴定。结果显示,本研究构建的IECs-cDNA文库其库容量为1.3×10~8 pfu/mL,插入cDNA片段长度为250～2500 bp,文库阳性率达100%。本研究结果表明构建的IECs-cDNA文库具有较好的多态性,为后续研究PEDV与宿主细胞蛋白之间的互作关系提供了有力支持。Swine small intestinal epithelial cells (IECs) are known as the target cells of Porcine epidemic diarrhea virus (PEDV). However, little is known about the interaction of PEDV and IECs. In this study, total RNA of IECs was extracted, reverse-transcribed into the corresponding complementary DNA (cDNA) by the SMART technology, and then co-transfected into the Y187 component cells with pGADT7-Rec and carrier DNA. The target library was screened by using the selective medium (SD/-Leu) and its quantity and quality were assessed using titration and PCR. The results showed that the titer of the constructed IECs-cDNA library was 1.3×108 pfu/mL and the length of the inserted cDNA ranged from 250 to 25 000 bp. These results suggested the IECs-cDNA library had a good polymorphism and could serve as a solid basis for further research on the interaction of PEDV and host cells.

关 键 词：猪流行性腹泻病毒 猪小肠上皮细胞 酵母双杂交cDNA文库 

分 类 号：S852.659.6[农业科学—基础兽医学]