| 重组pEGFP—C2-p100-TSN点突变质粒构建及表达 |
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| 引用本文： | 高星杰,邵洁,苏超,杨洁.重组pEGFP—C2-p100-TSN点突变质粒构建及表达[J].国外医学：分子生物学分册,2010(2):143-146. |
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| 作者姓名： | 高星杰 邵洁 苏超 杨洁 |
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| 作者单位： | [1]天津医科大学免疫学教研室,天津市300070 [2]国家教育部免疫微环境与疫病重点实验室,天津市300070 [3]天津市细胞与分子免疫学重点实验室,天津市300070 |
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| 基金项目： | 国家高技术研究发展计划（863计划）（No.2007AA02211.5）,国家自然科学基金（No.30670441,30970562,30670802,3081-1130394,30911130165,30970582）,国家重点基础研究发展规划（973计划）（No.2009CB918903）,天津市科委国际合作项目（N0.07JCZDJC07300）,国家自然科学重大研究计划培育项目（No.90919032）,国家教育部高等学校博士学科点专项科研基金（No.20091202110001）,天津市教委重点项目（No.2008ZD01） |
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| 摘 要： | 目的将6个不同的p100-TSN．Mutants基因片段分别定向连入PEGFP—C2质粒中，使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达，从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础。方法利用EcoR I和Xho I双酶切方法从6个pcDNA3．1（＋）-p100-TSN．Mutants重组质粒中分别获得p100-TSN．Mutants的cDNA片段，将其连人pEGFP—C2质粒载体中，再将成功构建的6个pEGFP—C2-p100-TSN．Mutants质粒分别转染COS7细胞中，荧光显微镜下观察绿色荧光蛋白表达。结果①将重组质粒进行双酶切鉴定可见p100-TSN．Mutants的cDNA片段；②转染重组质粒后可观察到绿色荧光蛋白的表达。结论①6个pEGFP—C2-p100-TSN．Mutants重组质粒构建成功；②p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达。
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| 关 键 词： | 人类P100蛋白 p100-TSN Mutants pEGFP—C2 重组质粒 融合蛋白 |
Construction and Expression of pEGFP-C2-p100-TSN. Mutants Recombinant Plasmids |
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| Authors: | GAO Xingjie SHAO Jie SU Chao YANG Jie |
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| Institution: | (Department of Immunology, Tianjin Medical University, Tianjin, 300070, China;2. Key Laboratory of Immunology Microenuironment and Disease, Ministry of Education of China, Tianjin, 300070, China;3. Tianjin Key Laboratory of Cellular and Molecular Immunology, Tianjin, 300070, China) |
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| Abstract: | Objective To construct eukaryotic green fluorescence protein expressing recombinant plasmids, pEGFP-C2-p100-TSN. Mutants, which contain human p100-TSN. Mutants cDNAs. Methods The p100-TSN. Mutants cDNAs were purified from EcoRI/XhoI digested pcDNA3.1 （ ＋ ） -p100-TSN. Mutants plasmids and inserted into pEGFP-C2 fluorescent expressing vector. These recombinant pEGFP-C2-p100-TSN. Mutants plasmids were transfected into COS7 cell line, and the expression of green fluorescent fusion proteins was observed by fluorescence microscope. Results ①The fragments of p100-TSN. Mutants were detected by restriction double enzyme digestion. ②The green fluorescent fusion proteins were observed in COS7 cell after the transfection. Conclusion ①The p100-TSN. Mutants fragments were correctly inserted into pEGFP-C2 vector. ② The recombinant pEGFP-p100-TSN. Mutants plasmids effectively expressed GFP-p100-TSN. mutants fusion protein. |
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| Keywords: | human p100 protein p100-TSN mutants pEGFP-C2 recombinant plasmid fusion protein |
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