与吕氏泰勒虫TlSP蛋白互作的绵羊淋巴细胞蛋白酵母双杂交系统的筛选  

Screening of proteins interacted with Theileria luwenshuni TlSP protein with ovine lymphocytes by yeast two-hybrid system

作　　者：秦鸽鸽[1,2] 韩元[1] 李有全[2] 殷宏[2,3] 罗建勋[2,3] 蔡葵蒸[1] 

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2016年第1期31-37,共7页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31272556);农业部肉牛牦牛项目(CARS-38);农业科技创新工程项目(ASTIP);国家农业公益性行业科研专项(201303035)

摘　　要：为研究吕氏泰勒虫入侵绵羊淋巴细胞的机理,用绵羊外周血淋巴细胞构建酵母双杂交文库,筛选与TlSP蛋白互作的蛋白。以密度梯度离心法分离出未感染泰勒虫的绵羊淋巴细胞,TRIzol法提取总RNA,建立酵母双杂交cDNA文库。结果显示,对文库进行评定,细胞密度为1.075×109/mL,插入片段长度在250~3 000bp之间,文库滴度为1.088×108 CFU,重组率为92%。利用诱饵质粒pGBKT7-TlSP对文库进行筛选,结果筛选到9种蛋白。生物信息学分析结果显示,这些蛋白主要参与调控基因转录、mRNA翻译、细胞的增殖、分化和凋亡等过程。上述结果表明,通过酵母双杂交技术筛选出与TlSP互作的蛋白,据此推测TlSP蛋白可能与吕氏泰勒虫在绵羊淋巴细胞内的增殖相关。The function of TlSP protein about invasion was studied through screening of the TlSP interacted proteins by the yeast two-hybrid system.The lymphocytes were isolated from uninfected sheep with protozoan by density gradient centrifuge,the total RNA was extracted from these cells by TRIzol reagent,and an yeast hybrid cDNA library was established.In result,the library evaluation shows that the cell density was 1.075×109 cell/mL,the insert fragments were between 250 bp and 3 000 bp and an average size of insert of the capacity of the library was 1.088×108 CFU with a recombinant rate of 92%.Nine proteins were obtained by using yeast two-hybrid system,and the bioinformatics analysis showed that these proteins were involved in regulating gene transcription,mRNA translation,cell proliferation,differentiation and apoptosis.In conclusion,the TISP interacted proteins screened by yeast two-hybrid may be involved in replication of T.luwenshuni in ovine lymphocyte.

关 键 词：吕氏泰勒虫 绵羊淋巴细胞 酵母双杂交 CDNA文库 

分 类 号：S852.723[农业科学—基础兽医学]