脑心肌炎病毒VP1蛋白酵母双杂交诱饵载体的构建及验证

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.030

生物技术通报 ›› 2015, Vol. 31 ›› Issue (1): 198-202.doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.030

脑心肌炎病毒VP1蛋白酵母双杂交诱饵载体的构建及验证

谢晶莹^1，3 冯若飞^1，2 徐雷³ 张海霞² 李向茸² 侯兰新³ 马忠仁²

（1.西北民族大学生物工程与技术国家民委重点实验室，兰州 730030；2.甘肃省动物细胞工程技术研究中心，兰州 730030；3.西北民族大学生命科学与工程学院，兰州 730030）

收稿日期:2014-09-26 出版日期:2015-01-09 发布日期:2015-01-10
作者简介:谢晶莹，女，硕士研究生，E-mail：xjy_1314@126.com
基金资助:
国家自然科学基金项目（31460665，31160033），教育部“长江学者和创新团队发展计划”项目（IRT13091），西北民族大学研究生科研创新项目（ycx13180）

Construction and Identification of Bait Vectors with VP1 Gene of Encephalomyocarditis Virus in Yeast Two-hybrid System

Xie Jingying^1，3, Feng Ruofei^1，2, Xu Lei³, Zhang Haixia², Li Xiangrong², Hou Lanxin³, Ma Zhongren²

（1. Key Bio-engineering and Technology Laboratory of the Northwest University for Nationalities，Lanzhou 730030；2.Gansu Engineering Research Center for Animal Cell，Lanzhou 730030；3. Life Science and Engineering College of Northwest University for Nationalities，Lanzhou 730030）

Received:2014-09-26 Published:2015-01-09 Online:2015-01-10

摘要/Abstract

摘要： 为筛选与脑心肌炎病毒VP1蛋白相互作用的靶细胞cDNA文库蛋白，构建VP1蛋白的诱饵载体pDHB1-VP1。扩增EMCV的VP1基因并克隆至pMD18-T载体中，经测序验证正确后定向克隆至酵母双杂交诱饵载体pDHB1。将重组pDHB1-VP1载体进行酶切验证和测序分析，并转化酵母报告菌株NMY51，检测其在酵母细胞中有无表达和自激活作用。结果表明，构建的pDHB1-VP1基因可以在酵母细胞中正确表达，产物大小约66 kD，而且可以与兔抗EMCV血清发生特异性结合，有较好免疫原性。成功构建了诱饵载体pDHB1-VP1，可以在酵母细胞中表达且其对报告基因无自激活作用，可以应用于酵母双杂交筛选试验中。

关键词: 脑心肌炎病毒, VP1蛋白, 酵母双杂交, 诱饵蛋白

Abstract: Bait vector pDHB1-VP1 was constructed for screening cellular proteins interacting with VP1 protein of encephalomyocarditis virus from yeast two-hybrid cDNA library of target cells in this study. VP1 gene was amplified and cloned into pMD18-T vector. After being verified by sequencing, it was directional cloning into bait vector pDHB1 of yeast two-hybrid system. Then the recombinant plasmid was identified by enzyme digestion and sequencing and transformed into yeast cells NMY51．The bait vectors’ expression and self-activation to reporter genes were tested．The results showed that the bait plasmid pDHB1-VP1 could express in yeast cells, and product size was 66 kD. It was specific binding with rabbit anti EMCV serum, which showed better immunogenicity. Bait plasmid pDHB1-VP1 was successfully constructed, could express in yeast cells and proved to be no self-activation to reporter genes．It could be used in the yeast two-hybrid system screening test．

Key words: encephalomyocarditis virus, vp1 protein, yeast two-hybrid, bait

谢晶莹,冯若飞,徐雷,张海霞,李向茸,侯兰新,马忠仁,. 脑心肌炎病毒VP1蛋白酵母双杂交诱饵载体的构建及验证[J]. 生物技术通报, 2015, 31(1): 198-202.

Xie Jingying, Feng Ruofei, Xu Lei, Zhang Haixia, Li Xiangrong, Hou Lanxin, Ma Zhongren. Construction and Identification of Bait Vectors with VP1 Gene of Encephalomyocarditis Virus in Yeast Two-hybrid System[J]. Biotechnology Bulletin, 2015, 31(1): 198-202.

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脑心肌炎病毒VP1蛋白酵母双杂交诱饵载体的构建及验证

Construction and Identification of Bait Vectors with VP1 Gene of Encephalomyocarditis Virus in Yeast Two-hybrid System

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