生物技术通报 ›› 2015, Vol. 31 ›› Issue (1): 131-137.doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.020

• 研究报告 • 上一篇    下一篇

绿色木霉TvALP-linker-TvRBL融合基因的构建与原核表达

徐杨玉1,2 温世杰 李海芬 李玲2 梁炫强   

  1. (1.广东省农业科学院作物研究所,广州 510640;2.华南师范大学生命科学学院,广州 510631)
  • 收稿日期:2014-05-22 出版日期:2015-01-09 发布日期:2015-01-10
  • 作者简介:徐杨玉,男,硕士,研究方向:生物防治微生物学;E-mail:574301612@qq.com
  • 基金资助:
    国家“863”计划项目(2006AA10Z156),广东省自然科学基金重点项目(07117967)

Construction and Prokaryotic Expression of Trichoderma viride TvALP-linker-TvRBL Fusion Gene

Xu Yangyu1,2, Wen Shijie, Li Haifen, Li Ling2, Liang Xuanqiang   

  1. (1. Crops Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640;2. College of Life Science,South China Normal University,Guangzhou 510631)
  • Received:2014-05-22 Published:2015-01-09 Online:2015-01-10

摘要: TvALP基因和TvRBL基因间加入一段柔性链接头(linker)基因序列,构建融合基因。生物信息学分析表明该融合基因含有一段信号肽序列。利用RT-PCR技术从绿色木霉菌丝体总RNA中扩增出TvRBL基因、TvALP基因和去除信号肽的?TvALP基因,分别克隆到原核表达载体pET30a,构建重组质粒pET30a-TvALP-linker-TvRBL和pET30a-?TvALP-linker-TvRBL,转化大肠杆菌BL21(DE3)pLysS,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达。SDS-PAGE电泳分析结果表明,去除信号肽的表达质粒pET30a-?TvALP-linker-TvRBL在大肠杆菌BL21(DE3)pLysS中获得了表达,在60 kD处有一条蛋白质特异条带,与预测的目的产物蛋白条带大小一致。

关键词: 绿色木霉, TvALP-linker-TvRBL融合基因, 信号肽, 原核表达

Abstract: Fusion gene were constructed by linking the TvALP and TvRBL genes with a flexible chain(linker). Bioinformatics analysis showed that the fusion gene contained a signal peptide. The TvRBL、TvALP and ?TvALP genes were cloned by RT-PCR from the total RNA of Trichoderma viride mycelium, which in turn were cloned into expression plasmid pET30a to construct prokaryotic expression plasmids pET30a-TvALP-linker-TvRBL and pET30a-?TvALP-linker-TvRBL, then these two expression plasmids were transformed into E.coli BL21(DE3)pLysS, and protein expression were induced by IPTG. SDS-PAGE was used to analyze the expression of the fusion protein. The result showed that expression plasmid pET30a-?TvALP-linker-TvRBL was obtained expression in E.coli BL21(DE3)pLysS, which was a specific band at about 60 kD in size and identical with the expected molecular weight of the fusion protein.

Key words: Trichoderma viride, TvALP-linker-TvRBL fusion gene, signal peptide, prokaryotic expression