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重组真核质粒pEGFP-C2-hG3BP-Domain(1～5)的构建及表达

朱梦瑜1,高星杰1,钱宝鑫1,杨洁2   

  1. 1. 天津医科大学基础医学院免疫教研室
    2. 天津医科大学免疫教研室
  • 收稿日期:2011-02-16 修回日期:2011-06-19 出版日期:2011-11-15 发布日期:2011-11-15
  • 通讯作者: 朱梦瑜

Construction and Expression of pEGFP-C2-hG3BP-Domain (1-5) Recombinant Eukaryotic Plasmids

  • Received:2011-02-16 Revised:2011-06-19 Published:2011-11-15 Online:2011-11-15
  • Contact: Meng-Yu ZHU

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摘要： 目的：将人类G3BP(Ras-GTPase Activating Protein SH3 Domain Binding Protein)蛋白Domain(1～5)基因片段分别定向连入pEGFP-C2质粒，使G3BP蛋白各功能片段与绿色荧光蛋白在HeLa细胞内融合表达。方法：以重组质粒pEGFP-C1-G3BP为模板，PCR法扩增出目的基因，利用EcoRI和BamHI双酶切法将目的片段连接到pEGFP-C2载体上，再将构建成功的pEGFP-C2-hG3BP-Domain(1～5)重组质粒转染入HeLa细胞内，以荧光显微镜及Western印迹法检测绿色荧光蛋白与目的蛋白的融合表达情况。结果：以单/双酶切及基因测序法鉴定构建的重组质粒均无误，荧光显微镜及Western印迹结果均检测到绿色融合蛋白的表达。 结论：重组pEGFP-C2-hG3BP-Domain(1～5)质粒成功构建并表达。

关键词: 人类G3BP蛋白, pEGFP-C2, 重组质粒, 融合蛋白

Abstract: Objective：To construct eukaryotic green fluorescent protein (GFP) expressing recombinant plasmids, pEGFP-C2-hG3BP-domain (1～5), which contain domain (1～5) fragments of human G3BP. Methods：The genes of G3BP fragments were amplified by PCR from the recombinant pEGFP-C1-G3BP plasmid and inserted into pEGFP-C2 fluorescent expressing vector with EcoRI and BamHI sites. These recombinant pEGFP-C2-hG3BP-Domain(1～5) plasmids were transfected into HeLa cells and the expression of green fluorescent fusion proteins was examined by fluorescence microscope and Western blotting assay. Results：The domain (1～5) fragments of G3BP were sequenced correctly and detected in the products of the restriction single/double enzyme digestion. The green fluorescent fusion proteins were also detected in the transfected HeLa cell by fluorescence microscope and Western blotting assay. Conclusion: These recombinant eukaryotic plasmids of pEGFP- C2-G3BP (1～5) were constructed successfully and expressed effectively.

Key words: Human G3BP protein, pEGFP-C2, Recombinant plasmid, Fusion protein