生物学杂志

• 研究报告 • 上一篇    下一篇

蛇床子三螺旋转录因子基因的克隆与表达分析

  

  1. 1. 安徽中医药大学 科研实验中心 新安医学教育部重点实验室, 合肥 230038; 
    2. 安徽中医药大学 药学院, 合肥 230038
  • 出版日期:2016-06-18 发布日期:2016-06-18
  • 通讯作者: 吴家文,教授,博士,研究方向为中药分子生物学,E-mail:wujiawen@ahtcm.edu.cn
  • 作者简介:王希强,硕士,研究方向为中药分子生物学,E-mail:xqwang547@163.com
  • 基金资助:
    安徽省自然科学基金项目(1608085MH177);安徽省高校优秀青年人才支持计划重点项目(gxyqZD2016139);安徽省2015年度留学人员科技活动择优资助项目;安徽中医药大学自然科学研究基金项目(2015zr017, 2015zr018);安徽中医药大学人才引进科研项目(2015RC002)

Cloning and expression analysis of trihelix transcription factor gene in Cnidium monnieri#br#

  1. 1. Key Laboratory of Xin′an Medicine, Ministry of Education; 
    2. School of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei 230038, China)
  • Online:2016-06-18 Published:2016-06-18

摘要: 研究的目的是克隆中药蛇床子[Cnidium monnieri (L.) Cuss.]三螺旋转录因子(Trihelix transcription factor,ttf)基因,诱导、鉴定其蛋白体外表达,并利用生物信息学方法分析该蛋白的结构和性质。主要采用RT-PCR方法扩增目的基因,连接到pMDTM19(Simple)-T克隆载体中,核酸序列鉴定正确后再将目的基因克隆到pET-22b(+)表达载体上,将获得的重组质粒转化到大肠杆菌E.coli BL21细胞中,以不同浓度的诱导剂诱导其表达,进一步利用聚丙烯酰胺凝胶电泳和免疫印迹方法检测目的蛋白的表达情况,并利用各种生物学软件对目的蛋白进行生物信息学分析。结果获得了ttf,构建了该基因的克隆和表达重组质粒,该基因在大肠杆菌BL21中能够被诱导表达,Western Blot结果表明,在预计的分子质量21 ku位置得到了目的蛋白;生物信息学分析表明,目的蛋白为可溶性蛋白,可能定位于细胞核和线粒体中;部分区域极有可能形成卷曲螺旋。研究首次成功体外克隆并在原核生物中表达了蛇床子三螺旋转录因子,并对其进行了系统的生物信息学分析,为进一步研究其结构和功能奠定了基础。

关键词: 蛇床子, 三螺旋转录因子, 基因克隆, 蛋白质表达, 生物信息学

Abstract:

The aim of this study is to clone the Trihelix transcription factor (ttf) gene in a Chinese
medicinal plant Cnidium monnieri(L.) Cuss. and express it in E.coli cells, followed by bioinformatic analysis.
The ttf cDNA was amplified by RT-PCR from Cnidium monnieri(L.)Cuss, and plasmid pMD19-T-ttf was constructed by
TA cloning. The cDNA fragment was subcloned to pET-22b(+), and recombinant plasmid pET-22b-ttf was verified
by restriction endonuclease analysis and DNA sequencing. The recombinant plasmid was transformed into E.coli
BL21, and pET-22b-ttf fusion protein was expressed with induction of IPTG at different concentrations, and
then illustrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Western-Blot assay
revealed that fusion protein could be identified by His-tag antibody. The fusion protein was about 21 ku as
expected. The molecular characteristics such as physiochemical properties, conserved domain, and sub-cellular
localization of the TTF protein were determined using a series of bioinformatic tools. ttf was cloned and
recombinant plasmid pET-22b-ttf was constructed, and then the fusion protein was expressed successfully, which
will lead to the further study of the structure and function of ttf.

Key words: