酵母双杂交诱饵蛋白日本血吸虫抱雌沟蛋白重组质粒的构建及鉴定  

CONSTRUCTION OF DNA-BD VECTOR WITH SJGCP FOR YEAST TWO-HYBRID SYSTEM

作　　者：吴启进[1,2] 金亚美[1] 李勉[3] 刘金明[1] 王小波[2] 石火英[2] 林矫矫[1] 

机构地区：[1]中国农业科学院上海兽医研究所国家防治动物血吸虫病专业实验室农业部动物寄生虫学重点开放实验室,上海200241 [2]扬州大学兽医学院,扬州225009 [3]河南农业大学动物医学学院,郑州450002

出　　处：《中国动物传染病学报》2010年第3期51-55,共5页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(30800819);上海市浦江人才计划(09PJ14118)

摘　　要：为筛选与血吸虫抱雌沟蛋白相互作用的因子,本研究以抱雌沟蛋白(gynecophoral canal protein ofSchistosoma japonicum,SjGCP)为诱饵蛋白构建了用于酵母双杂交筛选系统的重组质粒。根据日本血吸虫抱雌沟蛋白的基因序列设计引物,经过PCR扩增、酶切回收,与经相应酶切的线性化载体pBGKT7-BD连接构建重组质粒。随后将重组质粒pGBKT7-BD-SjGCP转化酵母菌株,测定融合蛋白BD-SjGCP在酵母菌中的自激活活性、对酵母菌生长的影响及在酵母中的表达情况。结果显示构建的重组诱饵蛋白质粒pGBKT7-BD-SjGCP能够在酵母细胞内正确表达,没有独自激活报告基因表达的活性,且其表达对酵母细胞没有毒性,不影响酵母细胞的生长。可见,重组质粒pGBKT7-BD-SjGCP可用于筛选与血吸虫抱雌沟蛋白相互作用的物质。Yeast two-hybrid system could be used to screen other proteins interacting with the gynecophoral canal protein of Schistosoma japonicum（SjGCP）.In this study,the recombinant plasmid pGBKT7-BD-SjGCP was constructed by inserting the target gene into vector pGBKT7-BD and then transformed into yeast cells AH109 and Y187.The autoactiviation of the BD-SjGCP fusion protein and the toxicity to yeast cells growth was examined.Results indicated that the recombinant fusion protein was expressed in yeast cells without interruption of cellular growth.In addition,the recombinant protein has no transcription activity of reporter genes or toxicity to the yeast cells.This study suggests the construction can be used for yeast two-hybrid systems.

关 键 词：酵母双杂交技术 抱雌沟蛋白 自激活 

分 类 号：S852.735[农业科学—基础兽医学] Q7[农业科学—兽医学]