丙型肝炎病毒核心蛋白酵母双杂交诱饵载体的构建及人胎肝cDNA文库的扩增、纯化和鉴定  

Construction of the bait vector of HCV core in yeast two-hybrid system and amplification,purification,evaluation of the cDNA gene bank from human fetal liver

作　　者：刘敏[1] 张伟[2] 陈天艳[1] 蔺淑梅[1] 赵良[1] 刘锦锋[1] 赵英仁[1] 张树林[1] 

机构地区：[1]西安交通大学医学院第一附属医院传染科,陕西西安710061 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032

出　　处：《西安交通大学学报（医学版）》2006年第1期20-22,45,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30471532);陕西省科学技术研究发展计划项目(No.2004K15-G1)

摘　　要：目的用含丙型肝炎病毒核心(HCV core)蛋白的cDNA片段构建酵母双杂交诱饵载体,并进行人胎肝cDNA文库的扩增、纯化和鉴定。方法PCR扩增含HCV core蛋白不同大小的cDNA片段,分别克隆入pUC19质粒,经测序正确后,再亚克隆入酵母双杂交诱饵载体pGBKT7中。扩增人胎肝cDNA文库并纯化、鉴定。结果获得含HCVcore蛋白的cDNA片段,并成功克隆入pGBKT7中。待转化的人肝cDNA文库滴度在5×108左右,纯化后的质粒DNA质量浓度约1 g/L。用EcoRⅠ、XhoⅠ双酶切显示插入片段大小不一。结论成功构建了核心蛋白的酵母双杂交诱饵载体;扩增、纯化的人胎肝cDNA文库的多样性很好,适合于筛选。为用酵母双杂交技术研究与HCV core蛋白相互作用的蛋白打下坚实基础。Objective To construct the bait vector of HCV core region in yeast two-hybrid system, and to amplify, purify and evaluate the cDNA gene bank of human fetal liver. Methods The cDNA fragments encoding HCV core region were amplified by PCR, and then were cloned into pUC19. After being verified by sequencing, they were subcloned into the bait vector pGBKT7 of yeast two-hybrid system, subsequently amplified and purified. And then the cDNA gene bank of human fetal liver was evaluated. Results The cDNA fragments of HCV core region were amplified successfully. The cDNA gene bank of human fetal fetal liver was about 5×10^8 ; the purified DNA was about 1 g/L; and the built in fragments were different in size after digested with EcoR Ⅰ and Xho Ⅰ. Conclusion The bait vector HCV core in yeast two-hybrid system 3 is constructed successfully. The diversity of cDNA of human fetal liver after amplified and purified is suitable for screening. These lay a rigid basis for further study of the HCV core protein and may help us in understanding how HCV works.

关 键 词：丙型肝炎病毒核心蛋白 酵母双杂交 CDNA文库 

分 类 号：R373.2[医药卫生—病原生物学]