摘要
目的:获得不包含C末端跨膜区的HCV包膜糖蛋白E2蛋白(E2 661)编码基因,构建酵母双杂交诱饵载体,并检测对报告基因的激活作用。方法:PCR扩增HCV E2661 cDNA片段,先克隆入pMD-18T载体中,经测序正确后,再亚克隆入诱饵载体 pGBKT7,将重组质粒pGBKT7-E2661用醋酸锂法转化入酵母菌AH109,检测目的蛋白在酵母细胞中的表达以及对报告基因有无激活作用。结果:成功构建含有HCVE2661基因片段的酵母双杂交诱饵载体,目的片段可在酵母细胞AH109中正确表达,对酵母菌无毒性且对报告基因无自主激活作用。结论:可利用所构建的pGBKT7-E2661作为酵母双杂交系统中的"诱饵",进行相互作用分子的筛选研究。
Objective： To construct the bait vector with the truncated HCV E2 （E2661） gene in yeast two- hybrid system and to investigate the effect of its expression on the growth of yeast cell. Method： HCV E2661 cDNA fragment was amplified by polymerase chain reaction（PCR）, and was subsequently cloned into pMD - 18T vector. After verification by sequencing, it was subcloned into the plasmid pGBKT7 of the yeast two - bybrid system. The recombinant plasmid pGBKT7 - E2661 was then transformed into yeast stain AH109. The expression product was assayed by westernblot, and the toxicity and autonomous activation of this recombinant plasmid in yeast cells were tested. Result： The bait vector with HCV E2 in yeast two - hybrid system was constructed successfully. The expression product of the fragment was expressed correctly and not toxic to AH109 and could not activate the reporter genes. Conclusion： The expression product of the plasmid pGBKT7- E2661 could be used as a bait to search for molecule interacting with the HCV E2 protein in yeast two- hybrid system.
出处
《生物技术》
CAS
CSCD
2008年第5期9-11,共3页
Biotechnology
基金
国家自然科学基金项目资助(No.30600021)
关键词
丙型肝炎病毒
包膜糖蛋白
酵母双杂交
诱饵载体
hepatitis C virus
envelope glycoprotein
yeast two- hybrid system
bait vector
