Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V

ZHONG Liang

doi:10.13406/j.cnki.cyxb.2008.03.001

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Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V

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- Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V
- Journal of Chongqing Medical University Issue 3, Pages: 257-260(2008)
- 作者机构：
  
  1. 重庆医科大学临床检验诊断学省部共建教育部重点实验室
  2. 重庆医科大学临床检验诊断学省部共建教育部重点实验室,重庆,400016
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2008.03.001
  CLC： Q78
- Published：2008，
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[1]钟梁,王翀,刘北忠,王东生,郝坡,刘畅,金丹婷,王春光.酵母双杂交诱饵载体pGBKT7-PML-V的构建、表达及鉴定[J].重庆医科大学学报,2008(03):257-260.

ZHONG Liang. Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V. [J]. Journal of Chongqing Medical University (3):257-260(2008)
[1]钟梁,王翀,刘北忠,王东生,郝坡,刘畅,金丹婷,王春光.酵母双杂交诱饵载体pGBKT7-PML-V的构建、表达及鉴定[J].重庆医科大学学报,2008(03):257-260. DOI： 10.13406/j.cnki.cyxb.2008.03.001.

ZHONG Liang. Construction,expression and identification of yeast two-hybrid bait vector pGBKT7-PML-V. [J]. Journal of Chongqing Medical University (3):257-260(2008) DOI： 10.13406/j.cnki.cyxb.2008.03.001.

摘要

目的:构建早幼粒细胞白血病基因变异蛋白（Promyelocytic leukemia

PML-V）的诱饵表达载体

为应用酵母双杂交系统（Yeast two-hybrid system）筛选与PML-V相互作用的蛋白建立实验基础。方法:PCR扩增PML-V

克隆入诱饵载体pGBKT7中

经测序鉴定后

将构建好的诱饵载体pGBKT7-PML-V转化到酵母细胞AH109中

并利用蛋白印迹法（Western Blotting）分析诱饵蛋白的表达情况。同时检测诱饵蛋白有无毒性、渗漏和自激活作用。结果:成功扩增了PML-V基因片断

并克隆入pGBKT7中

测序结果正确。诱饵载体成功转化到酵母细胞AH109中

诱饵蛋白无毒性、渗漏和自激活作用

Western Blotting分析证实酵母细胞表达诱饵蛋白。结论:成功构建了PML-V结合域的酵母诱饵表达载体

为进一步运用酵母双杂交技术筛选与之相互作用的蛋白奠定了基础。

Abstract

Objective:To construct the bait expression vector pGBKT7-PML-V of retinoic acid receptor variant protein

for screening the target proteins interacting with the bait protein through the yeast two-hybrid system.Methods:The fragments of PML-V binding domain was amplified by PCR

and then was cloned into the bait expression vector pGBKT7.After being verified by sequencing

the bait vector pGBKT7-PML-V was transformed into AH109 yeast cells.Then the expression of the bait protein was analyzed by Western Blotting.Toxicity

leakage and serf-activation of the bait protein were detected.Results: PML-V was amplified and cloned into pGBKT7 successfully.The bait vector was transformed into AH109 as well and no toxicity and self-activation were found.The expression of the bait protein was confirmed by Western Blotting.Conclusion:The bait expression vector of PML-V was constructed successfully

which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.

关键词

PML变异蛋白质粒构建诱饵蛋白

Keywords

PML-VVector constructionBait protein

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