吉林大学学报(医学版)

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RUNX3全长和不同结构域原核表达载体的构建及其重组蛋白表达

宋艳艳,王桂玲   

  1. (中国医科大学细胞生物学教研室 卫生部细胞生物学重点实验室,辽宁 沈阳 110001)
  • 收稿日期:2012-11-09 发布日期:2013-11-28
  • 通讯作者: 王桂玲 E-mail:(Tel:024-23256666-5347,E-mail:wanggl2000@163.com)
  • 作者简介:宋艳艳(1987-),女,内蒙古自治区通辽市人,在读医学硕士,主要从事肿瘤信号转导方面的研究。
  • 基金资助:

    国家自然科学基金资助课题(30871294);辽宁省自然科学基金资助课题(201102277)

Construction of prokaryotic expression vector of overall length and different domains of RUNX3 and expressions of their recombinant proteins

SONG Yan-yan,WANG Gui-ling   


  1. (Key Laboratory of Cell Biology,Ministry of Health,Department of Cell Biology,China Medical University,Shenyang 110001,China)
  • Received:2012-11-09 Published:2013-11-28

摘要:

目的:构建RUNX3全长和不同结构域的原核表达载体并表达其重组蛋白。方法:以pcDNA3.1-myc-RUNX3为模板,采用PCR法扩增RUNX3全长和各个结构域片段,通过EcoRⅠ和BamHⅠ酶切位点将RUNX3全长和不同结构域定向插入谷胱甘肽转移酶(GST)融合表达载体pGEX-4T-2中,在大肠杆菌(E.coli) BL21中诱导其融合蛋白表达,SDS-PAGE电泳检测其蛋白表达情况。结果:EcoRⅠ和BamHⅠ双酶切后得到与预期大小相符的FL(1 245 bp)、ΔRunt(843
 bp)、Nter(159 bp)、Runt(402 bp)和Cter(684 bp)5个载体片段。SDS-PAGE电泳检测,RUNX3全长及各个结构域截短GST-RUNX3截短融合蛋白(GST-FL、GST-Runt、GST-Nter和GST-Cter)相对分子质量分别为72 000、40 000、32 000和51 000。 结论:成功构建RUNX3全长和各个结构域截短的GST标签的原核表达载体,并实现其重组蛋白在原核细胞中的表达。

关键词: RUNX3, 载体, 蛋白诱导, 蛋白表达

Abstract:

Abstract:Objective
To construct the prokaryotic expression vectors of overall length and different domains of RUNX3 and to identify the expressions of their recombinant proteins.Methods The coding sequence of overall length and different domain truncations of RUNX3 were amplified from the pcDNA3.1-myc-RUNX3 plasmid  by PCR method and inserted into glutathione transferase(GST) fusion expression vector  pGEX-4T-2 through EcoRⅠand BamHⅠ restriction enzyme sites.Then they were transformed into E.coli BL21 and the fusion proteins were induced  and identified by SDS-PAGE electrophoresis.Results The vector fragments FL (1 245 bp),ΔRunt(843 bp),Nter(159 bp),Runt(402 bp), and Cter (684 bp) which were  consistent with the expected fragments were obtained after double enzyme digestion of EcoRⅠand BamHⅠ.The SDS-PAGE electrophoresis result showed that the relative molecular mass of GST-RUNX3 truncated fusion proteins of overall length and different domains of  RUNX3 (GST-FL,GST-Runt,GST-Nter and GST-Cter)were 72 000,40 000,32 000, and 51 000,respectively.Conclusion GST-tagged prokaryotic expression vectors overall length and   different truncated regions of RUNX3  are constructed  and their recombinant proteins are expressed successfully.

Key words: RUNX3, vector, inducing of protein, expression of protein

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