大黄鱼Wap65-2基因成熟肽区酵母双杂交诱饵载体的构建及鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.016

生物技术通报 ›› 2015, Vol. 31 ›› Issue (8): 108-113.doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.016

大黄鱼Wap65-2基因成熟肽区酵母双杂交诱饵载体的构建及鉴定

裘天休^{1, 2}, 李长红², 陈炯²

1. 宁波大学科技学院,宁波 315211; 2. 宁波大学海洋学院生物化学与分子生物学实验室,宁波 315211

收稿日期:2014-12-01 出版日期:2015-08-21 发布日期:2015-08-22
作者简介:裘天休,女,研究方向：鱼类分子免疫学;E-mail：qt618@163.com
基金资助:
国家自然科学基金项目（31402333）,高等学校博士学科点专项科研基金项目（20133305120008）,宁波市自然科学基金项目（2013A610160）,浙江省教育厅科研项目（理）（Y201327619）

Construction and Identification of a Bait Vector for the Mature Peptide of Wap65-2 of Large Yellow Croaker in Yeast Two-Hybrid System

Qiu Tianxiu^{1, 2}, Li Changhong², Chen Jiong²

1. College of Science and Technology,Ningbo University,Ningbo 315211; 2. Biochemistry and Molecular Biology Laboratory,School of Marine Science,Ningbo University,Ningbo 315211

Received:2014-12-01 Published:2015-08-21 Online:2015-08-22

摘要/Abstract

摘要： 用大黄鱼Wap65-2基因成熟肽区构建酵母双杂交诱饵质粒,并对诱饵质粒进行自激活活性及毒性检测。从大黄鱼（Larimichthys crocea）肝组织cDNA中扩增Wap65-2基因成熟肽片段（LcWap65-2m）,将其克隆至pGBKT7载体中,获得诱饵载体pGBKT7-LcWap65-2m,经PCR、酶切和测序验证无误后,转化至酵母菌株Y187中,在营养缺陷培养基中观察重组蛋白的毒性作用和自激活活性,同时利用Western blot分析重组蛋白的表达。成功扩增LcWap65-2m,并克隆至pGBKT7载体中;Western blot检测结果表明,pGBKT7-LcWap65-2m在酵母细胞中表达诱饵蛋白LcWap65-2m;表型筛选结果显示,LcWap65-2m无毒性作用和自激活活性。成功构建了LcWap65-2m酵母双杂交诱饵载体,为进一步利用酵母双杂交技术筛选与Wap65-2相互作用的蛋白奠定基础。

关键词: Wap65-2, 酵母双杂交, 表达, 自激活, 毒性

Abstract: The mature peptide of Wap65-2 gene（LcWap65-2m）in large yellow croaker（Larimichthys crocea）was used to construct the yeast two-hybrid bait vector, and its self-activation and toxic effect were measured. LcWap65-2m from the liver cDNA of large yellow croaker was amplified, and cloned into the plasmid pGBKT7. After verification by PCR amplification, enzyme digestion and sequencing, the bait vector pGBKT7-LcWap65-2m was transformed into the yeast strain Y187. The self-activation and toxic effect of the recombinant protein were detected in selective nutrition medium, and the expression of the bait protein LcWap65-2m was analyzed by Western blot. LcWap65-2m was successfully amplified and inserted into the plasmid vector pGBKT7. Western blot analysis showed that the bait protein LcWap65-2m was expressed in yeast cells;phenotypic screening tests revealed that the bait protein LcWap65-2m had no self-activation activity and toxic effect. In conclusion, the yeast two-hybrid bait vector pGBKT7-LcWap65-2m was successfully constructed, which laid the foundation for screening the protein interacted with the bait protein Wap65-2 using the yeast two-hybrid system.

Key words: Wap65-2, yeast two-hybrid, expression, self-activation, toxic effect

裘天休, 李长红, 陈炯. 大黄鱼Wap65-2基因成熟肽区酵母双杂交诱饵载体的构建及鉴定[J]. 生物技术通报, 2015, 31(8): 108-113.

Qiu Tianxiu, Li Changhong, Chen Jiong. Construction and Identification of a Bait Vector for the Mature Peptide of Wap65-2 of Large Yellow Croaker in Yeast Two-Hybrid System[J]. Biotechnology Bulletin, 2015, 31(8): 108-113.

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大黄鱼Wap65-2基因成熟肽区酵母双杂交诱饵载体的构建及鉴定

Construction and Identification of a Bait Vector for the Mature Peptide of Wap65-2 of Large Yellow Croaker in Yeast Two-Hybrid System

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