猪小肠上皮细胞酵母双杂交cDNA文库的构建及鉴定  

Construction and Identification of Yeast Two-hybrid cDNA Library of Porcine Intestinal Epithelial Cells

作　　者：徐天[1] 石达[1] 陈建飞[1] 谷凤丽[1,2] 时洪艳[1] 张鑫[1] 冯力[1,2] 

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 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《中国畜牧兽医》2014年第10期33-37,共5页China Animal Husbandry & Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项(0302013002);猪流行性腹泻病毒纤突蛋白与宿主蛋白相互作用机制研究;国家自然基金项目“猪流行性腹泻病毒N蛋白核定位特性及其对宿主细胞周期影响”(31172350)

摘　　要：为了更系统的研究病毒在猪肠道内侵入及致病的分子机制,本试验以猪小肠上皮细胞（intestinal epithelial cells,IEC）为模型,应用RNeasy Min Kit从猪IEC中提取细胞总RNA,反转录后,利用SMART技术合成双链cDNA（ds-cDNA）,通过同源重组的方法构建猪IEC酵母双杂交cDNA文库。结果显示,文库的滴度为8.4×108 CFU/mL,插入的ds-cDNA片段大小为0.5~2.0kb,平均长度约为1.1kb,文库重组率为100%。此文库为筛选与猪腹泻病毒相互作用的宿主蛋白及进一步阐明病毒的致病机制奠定了基础,为研制有效的药物或疫苗提供了依据。In order to study the molecular mechanism of invasion and virulence of the swine intestinal virus systematically,we constructed a porcine intestinal epithelial cells（IEC）cDNA yeast two-hybrid library.Taking advantage of RNeasy Min Kit,total cellular RNA was extracted from cultured porcine IEC,after reverse transcription,synthetise double stranded cDNA by the SMART technology,porcine IEC yeast cDNA library were constructed with homologous recombination.The results showed,library titer was 8.4×108 CFU/mL,the inserted fragment of ds-cDNA was 0.5to 2.0kb,the average length up to1.1kb with a high library recombination rate.This library could be used for screening intestinal host proteins which were interacted with virus proteins,provide reference for the further study of the pathogenesis of the virus,and provide basement for the development of effective drug or vaccine.

关 键 词：猪腹泻病毒 猪小肠上皮细胞 酵母双杂交 CDNA文库 

分 类 号：Q78[生物学—分子生物学]