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| TBK1相关激酶活性缺失突变体和泛素样结构域突变体的构建、表达及生物活性鉴定 | |
| 引用本文： | 魏从文,管楷,岳翔,袁媛,宋婷,郑子瑞,张艳红,钟辉.TBK1相关激酶活性缺失突变体和泛素样结构域突变体的构建、表达及生物活性鉴定[J].生物技术通讯,2011,22(5):671-674. |
| 作者姓名： | 魏从文 管楷 岳翔 袁媛 宋婷 郑子瑞 张艳红 钟辉 |
| 作者单位： | 1. 军事医学科学院生物工程研究所,北京,100850 2. 军事医学科学院生物工程研究所,北京100850;安徽大学生命科学院,安徽合肥230039 3. 军事医学科学院生物工程研究所,北京100850;吉林大学分子酶学工程实验室,吉林长春130012 |
| 摘 要： | 目的:构建TANK结合激酶1(TBK1)相关激酶活性缺失突变体和泛素样结构域突变体真核表达载体,检测该基因相关突变体在293细胞中的表达,并利用萤光素酶报告基因实验检测其生物活性。方法:根据文献报道的突变序列及QuickChange Site-Directed Mutagenesis实验设计手册,设计合成2条针对TBK1相关激酶活性缺失突变体和泛素样结构域突变体的引物,以实验室之前构建的TBK1野生型真核表达载体为模板,构建TBK1激酶活性缺失突变体和泛素样结构域突变体真核表达载体,分别命名为pcDNA3-Flag-TBK1(KD)、pcDNA3-Flag-TBK1(ΔULD)。以LipofactAMINE2000转染试剂转染至293细胞中进行瞬时表达,利用萤光素酶实验检测2种TBK1突变体诱导β干扰素(IFN-β)转录的情况。结果:测序结果表明,TBK1相关激酶活性缺失突变体和泛素样结构域缺失突变体真核表达载体构建成功,Western印迹检测表明其在293细胞中获得有效表达;用萤光素酶报告基因实验检测,与野生型TBK1相比,其相关激酶活性缺失突变体和泛素样结构域缺失突变体诱导IFN-β转录激活的作用明显降低。结论:真核表达的TBK1相关激酶活性缺失突变体和泛素样结构域突变体具有相应的生物学活性,为研究其功能奠定了基础。 |
| 关 键 词： | TANK结合激酶1 激酶活性缺失突变体 泛素样结构域突变体 真核表达 转录活性 |
Construction and Expression of TBK1 Kinasedead Mutant and Ubiquitin-like Domain Mutant in 293 Cell Lines and Identification of theirs Biological Activity |
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| WEI Cong-Wen,GUA N Kai,YUE Xiang,YUAN Yuan,SONG Ting,ZHENG Zi-Rui,ZHANG Yan-Hon,ZHONG Hui.Construction and Expression of TBK1 Kinasedead Mutant and Ubiquitin-like Domain Mutant in 293 Cell Lines and Identification of theirs Biological Activity[J].Letters in Biotechnology,2011,22(5):671-674. | |
| Authors: | WEI Cong-Wen GUA N Kai YUE Xiang YUAN Yuan SONG Ting ZHENG Zi-Rui ZHANG Yan-Hon ZHONG Hui |
| Institution: | WEI Cong-Wen 1,GUAN Kai 1,2,YUE Xiang 1,YUAN Yuan 1,SONG Ting 1,ZHENG Zi-Rui 1,3,ZHANG Yan-Hong 1,ZHONG Hui 1 1.Beijing Institute of Biotechnology,Beijing 100850,2.College of Life Science,Anhui University,Hefei 230039,3.Key Laboratory for Molecular Enzymology and Engineering,Jinlin University,Changchun 130012,China |
| Abstract: | Objective: To construct the TANK binding kinase-1(TBK1) kinasedead mutant and ubiquitin-like do-main mutant eukaryotic expressing plasmid,express them in 293 cell lines and identify theirs biological activities.Methods: According to the reported TBK1 mutant sequence and QuickChange Site-Directed Mutagenesis kit,two of specific DNA sequences were designed and synthesized.Using the plasmid of TBK1 which was constructed by re-searchers in our laboratory as templates,construct TBK1 kinasedead mutant and ubiquit... |
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