Screening of proteins interacting with HOXA11 by yeast two-hybrid system

Cheng Zhi; Gao Rui; Ma Linqiang; Huang Huizhe

doi:10.13406/j.cnki.cyxb.000756

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Screening of proteins interacting with HOXA11 by yeast two-hybrid system

更新时间：2024-01-17

- Screening of proteins interacting with HOXA11 by yeast two-hybrid system
- Journal of Chongqing Medical University Vol. 41, Issue 5, Pages: 443-447(2016)
- 作者机构：
  
  1. 重庆医科大学基础医学院细胞生物学与遗传学教研室
  2. 重庆医科大学发育生物学研究室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.000756
  CLC： R737.11
- Published：2016，
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[1]程志,高蕊,马林强,黄慧哲.利用酵母双杂交技术筛选与转录因子HOXA11相互作用蛋白[J].重庆医科大学学报,2016,41(05):443-447.

Cheng Zhi, Gao Rui, Ma Linqiang, et al. Screening of proteins interacting with HOXA11 by yeast two-hybrid system. [J]. Journal of Chongqing Medical University 41(5):443-447(2016)
[1]程志,高蕊,马林强,黄慧哲.利用酵母双杂交技术筛选与转录因子HOXA11相互作用蛋白[J].重庆医科大学学报,2016,41(05):443-447. DOI： 10.13406/j.cnki.cyxb.000756.

Cheng Zhi, Gao Rui, Ma Linqiang, et al. Screening of proteins interacting with HOXA11 by yeast two-hybrid system. [J]. Journal of Chongqing Medical University 41(5):443-447(2016) DOI： 10.13406/j.cnki.cyxb.000756.

摘要

目的:利用酵母双杂交技术从均一化的人肾透明细胞腺癌c DNA文库中筛选与转录因子HOXA11相互作用的蛋白。方法:培养人肾透明细胞腺癌细胞

提取RNA

采用SMART技术制备均一化的人肾透明细胞腺癌c DNA文库。同时

聚合酶链反应扩增人HOXA11基因片段（1

46 bp）

将此基因片段重组入p GBKT7载体

构建诱饵质粒p GBKT7-HOXA11

经酶切和测序验证质粒构建正确。将诱饵质粒转化酵母菌AH109

检测其有无毒性与自激活作用。随后

利用酵母双杂交系统从c DNA文库中筛选与HOXA11相互作用的蛋白。对筛选到的阳性克隆进行回转验证以及测序分析。结果:成功构建了人肾透明细胞腺癌c DNA文库和p GBKT7-HOXA11诱饵质粒。在AH109酵母细胞中

该质粒无毒性、无自激活作用。酵母双杂交筛选获得了5个与HOXA11相互作用阳性克隆。结论:获得人肾透明细胞腺癌细胞中与HOXA11相互作用蛋白

为进一步研究HOXA11在个体发育以及癌症发生发展中的作用机制奠定了基础。

Abstract

Objective:To screen proteins binding with HOXA11 from human renal clear cell adenocarcinoma c DNA library by yeast two-hybrid system. Methods:The RNA of human renal clear cell adenocarcinoma cell was obtained and then transformed into c DNA library using SMART technology. Simultaneously

the c DNA fragments of HOXA11(1-846 bp)were amplified by polymerase chain reaction and constructed into p GBKT7 vector as the bait plasmid. The bait plasmid was transferred into yeast AH109 strain. Its toxicity and autoactivation were tested. Subsequently

the c DNA library was screened with p GBKT7-HOXA11 as bait plasmid by yeast twohybrid system. Finally

the positive clones were sequenced and further analyzed. Results:Human renal clear cell adenocarcinoma c DNA library and bait plasmid p GBKT7-HOXA11 were constructed successfully. The bait plasmid has no toxicity or autoactivation in AH109.Five positive clones were obtained and validated. Conclusion:Proteins deduced to interact with HOXA11 are identified through yeast two-hybrid screening. These results help to elucidate the possible roles of HOXA11 in carcinogenesis and development.

关键词

HOXA11SMART技术酵母双杂交

Keywords

HOXA11SMART technologyyeast two-hybrid

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