禽呼肠孤病毒σA基因酵母双杂交诱饵载体的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2017.03.032

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (3): 847-853.doi: 10.16431/j.cnki.1671-7236.2017.03.032

禽呼肠孤病毒σA基因酵母双杂交诱饵载体的构建与鉴定

卢恒¹, 谢芝勋², 谢丽基², 黄莉², 黄娇玲², 王盛², 张艳芳², 曾婷婷², 范晴², 罗思思², 谢志勤², 邓显文²

1. 广西大学动物科学技术学院, 南宁 530004;
2. 广西壮族自治区兽医研究所, 广西兽医生物技术重点实验室, 南宁 530001

收稿日期:2016-09-06 出版日期:2017-03-20 发布日期:2017-03-21
通讯作者: 谢芝勋 E-mail:xiezhixun@126.com
作者简介:卢恒(1992-),男,河南郑州人,硕士,研究方向:兽医生物技术学,Tel:0771-3120371;E-mail:henglu2000@163.com
基金资助:
国家自然科学基金项目（31660715、31160512）；国家"万人计划"领军人材专项（2016-37）；广西特聘专家专项（2011B020）

Construction and Identification of Bait Vector of Avian Reovirus σA Gene in Yeast Two-hybrid System

LU Heng¹, XIE Zhi-xun², XIE Li-ji², HUANG Li², HUANG Jiao-ling², WANG Sheng², ZHANG Yan-fang², ZENG Ting-ting², FAN Qing², LUO Si-si², XIE Zhi-qin², DENG Xian-wen²

1. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
2. Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning 530001, China

Received:2016-09-06 Online:2017-03-20 Published:2017-03-21

摘要/Abstract

摘要：

为了筛选与禽呼孤病毒σA基因相互作用的宿主蛋白，本试验应用酵母双杂交技术构建禽呼孤病毒σA基因的诱饵载体pGBKT7-σA。从禽呼肠孤病毒S1133标准毒株抽提RNA，采用RT-PCR方法扩增得到σA基因片段，将其连接到诱饵载体pGBKT7上，把通过测序的重组诱饵载体命名为pGBKT7-σA。将重组诱饵载体转化酿酒酵母Y2HGold后，把不同浓度的诱饵载体转化液涂布在营养缺陷型培养基上，观察该重组诱饵载体在酵母细胞中有无毒性作用和自激活现象。结果显示，酵母双杂交诱饵载体pGBKT7-σA构建成功，且对酿酒酵母无毒性和自激活现象。本研究为进一步利用酵母双杂交技术筛选与σA蛋白互作的宿主蛋白奠定了基础。

关键词: 禽呼肠孤病毒; σA基因; 酵母双杂交; 诱饵载体

Abstract:

In order to screen the host protein that interacted with σA gene of avian reovirus, the bait vector pGBKT7-σA of avian reovirus σA gene in yeast two-hybrid system was constructed. Total RNA of avian reovirus S1133 standard strain was purified by using Trizol reagents. Avian reovirus σA gene was amplified by RT-PCR and was cloned into yeast two-hybrid bait vector pGBKT7. The recombinant bait vector was named as pGBKT7-σA. The recombinant bait vector was transformed into Saccharomyces cerevisiae Y2HGold. And then the recombinant bait vector of different concentrations was applied to the nutrient deficient medium. The results showed that the yeast two-hybrid bait vector had been constructed successfully, and the recombinant bait vector pGBKT7-σA on yeast cells had no toxicity and self-activation. This study laid a foundation for further screening host protein interacted with σA protein by yeast two-hybrid technique.

Key words: avian reovirus; σA gene; yeast two-hybrid system; bait vector

中图分类号:

S852.65

卢恒, 谢芝勋, 谢丽基, 黄莉, 黄娇玲, 王盛, 张艳芳, 曾婷婷, 范晴, 罗思思, 谢志勤, 邓显文. 禽呼肠孤病毒σA基因酵母双杂交诱饵载体的构建与鉴定[J]. 《中国畜牧兽医》, 2017, 44(3): 847-853.

LU Heng, XIE Zhi-xun, XIE Li-ji, HUANG Li, HUANG Jiao-ling, WANG Sheng, ZHANG Yan-fang, ZENG Ting-ting, FAN Qing, LUO Si-si, XIE Zhi-qin, DENG Xian-wen. Construction and Identification of Bait Vector of Avian Reovirus σA Gene in Yeast Two-hybrid System[J]. , 2017, 44(3): 847-853.

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禽呼肠孤病毒σA基因酵母双杂交诱饵载体的构建与鉴定

Construction and Identification of Bait Vector of Avian Reovirus σA Gene in Yeast Two-hybrid System

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