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Tudor-SN蛋白TSN结构域亚结构片段重组质粒的构建与表达

钱宝鑫1,高星杰2,朱梦瑜1,杨洁3   

  1. 1. 天津医科大学基础医学院免疫教研室
    2. 天津医科大学基础医学研究中心
    3. 天津医科大学免疫教研室
  • 收稿日期:2011-02-16 修回日期:2011-04-26 出版日期:2011-10-15 发布日期:2011-10-15
  • 通讯作者: 钱宝鑫

Construction and Determination of Recombinant Eukaryotic Plasmids of Tudor-SN Protein TSN Domain’s sub-Fragments

  • Received:2011-02-16 Revised:2011-04-26 Published:2011-10-15 Online:2011-10-15
  • Contact: Bao-Xin QIAN

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摘要： 摘要 目的：将人类Tudor-SN蛋白TSN结构域基因片段的4个亚片段分别定向连入pEGFP-C2载体的多克隆位点中，使它们能够分别与绿色荧光蛋白融合并在HeLa细胞中表达，为进一步研究Tudor-SN蛋白TSN结构域不同亚区的功能奠定基础。方法：以重组质粒pSG5-Tudor-SN-flag为模板，PCR扩增出目的基因，将双酶切后带有粘性末端的目的片段和pEGFP-C2载体连接，从而构建重组质粒pEGFP-C2-Tudor-SN-TSN（Ⅰ~Ⅳ）。将构建成功的重组质粒脂质体法转染HeLa细胞，并在荧光显微镜下观察融合蛋白的荧光表达情况，Western印迹检测融合蛋白的表达。结果：①对重组质粒进行双酶切鉴定可见Tudor-SN-TSN（Ⅰ~Ⅳ）的cDNA片段；②脂质体转染重组质粒后可观察到绿色荧光蛋白的表达。③Western印迹后可在相应位置检测到融合蛋白pEGFP-C2-Tudor-SN-TSN（Ⅰ~Ⅳ）。结论：①pEGFP-C2-Tudor-SN-TSN（Ⅰ~Ⅳ）重组质粒构建成功； ②目的片段可与绿色荧光蛋白在HeLa细胞中融合表达，融合蛋白可与抗GFP抗体结合用于蛋白检测，为深入研究Tudor-SN蛋白TSN结构域中不同亚结构片段的功能奠定基础。

关键词: 人类Tudor-SN蛋白, SND1, pEGFP-C2, 重组质粒, 融合蛋白

Abstract: Abstract Objective:To construct eukaryotic green fluorescent protein(GFP) expressing recombinant plasmids, pEGFP-C2-Tudor-SN-TSN(Ⅰ~Ⅳ), which contain 4 sub-fragments of human Tudor-SN TSN domain, respectively. Methods: The genes of Tudor-SN-TSN fragments were amplified by PCR from the recombinant pSG5-Tudor-SN-flag plasmid then the EcoRI/Sal I digested fragments were combined with pEGFP-C2 vector. The recombinant expression plasmids pEGFP-C2-TSN(Ⅰ~Ⅳ) were transfected into HeLa cells. The expression of fusion proteins were examined by fluorescent microscopy and Western blot. Results: (1) The sub-fragments of TSN domain can be detected in the products of the restriction double enzyme digestion; (2) The green fluorescent fusion proteins were observed in HeLa cells after transfection; (3) The fusion proteins pEGFP-C2-TSN(Ⅰ~Ⅳ) can be detected from the lysate of transfected HeLa cells by Western blot. Conclusion: (1) The expression plasmids pEGFP-C2-TSN(Ⅰ~Ⅳ) was successfully constructed. (2) These recombinant eukaryotic plasmids can be used for protein detection by binding with anti-GFP antibody for the further study on molecular mechanism in biologic function of Tudor-SN-TSN.

Key words: Human Tudor-SN protein, SND1, pEGFP-C2, Recombinant plasmid, Fusion protein