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| 人TBK1小干扰RNA质粒的构建及稳定干扰TBK1细胞株的筛选 | |
| 引用本文： | 孙静,魏从文,管楷,程孝中,徐扬,袁媛,尹晴晴,宋婷,郑子瑞,张艳红,钟辉.人TBK1小干扰RNA质粒的构建及稳定干扰TBK1细胞株的筛选[J].生物技术通讯,2010,21(2):163-167. |
| 作者姓名： | 孙静 魏从文 管楷 程孝中 徐扬 袁媛 尹晴晴 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;北京市公安医院,北京,100121 2. 军事医学科学院,生物工程研究所,北京,100850 3. 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 4. 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:构建人TANK结合激酶1(TBK1)的小干扰RNA真核表达质粒,并筛选出稳定干扰TBK1的细胞株。方法:根据文献报道的序列和载体的黏性末端,设计合成2条针对TBK1的DNA序列,退火后连接到载体pSUPER.retro.neo+gfp上,经测序分析正确后得到质粒psiTBK1。用脂质体转染质粒psiTBK1到MCF-7细胞中,经G418加压筛选稳定表达TBK1小干扰RNA的细胞株,用免疫印迹检测细胞中TBK1的表达情况,将干扰效果好的细胞株命名为MCF-7/siTBK1,再用萤光素酶试验检测MCF-7/siTBK1细胞对外源TBK1诱导干扰素-β(IFN-β)转录的情况。结果:免疫印迹结果证实建立的稳定细胞株MCF-7/siTBK1能够有效干扰TBK1的表达,并在外源TBK1存在的情况下抑制IFN-β的转录活性。结论:构建了表达TBK1小干扰RNA的质粒psiTBK1,筛选出稳定干扰TBK1表达的细胞株MCF-7/siTBK1,为深入研究TBK1在先天免疫中的作用提供了平台。 |
| 关 键 词： | TANK结合激酶1 小干扰RNA 萤光素酶试验 转录活性 |
Construction of the Small Interference RNA Expression Vector and Screening of the Stable Knockdown Cell Lines of Human TBK1 |
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| SUN Jing,WEI Cong-Wen,GUAN Kai,CHENG Xiao-Zhong,XU Yang,YUAN Yuan,YIN Qing-Qing,SONG Ting,ZHENG Zi-Rui,ZHANG Yan-Hong,ZHONG Hui.Construction of the Small Interference RNA Expression Vector and Screening of the Stable Knockdown Cell Lines of Human TBK1[J].Letters in Biotechnology,2010,21(2):163-167. | |
| Authors: | SUN Jing WEI Cong-Wen GUAN Kai CHENG Xiao-Zhong XU Yang YUAN Yuan YIN Qing-Qing SONG Ting ZHENG Zi-Rui ZHANG Yan-Hong ZHONG Hui |
| Institution: | SUN Jing1,2,WEI Cong-Wen1,GUAN Kai1,3,CHENG Xiao-Zhong1,XU Yang1,4,YUAN Yuan1,YIN Qing-Qing1,SONG Ting1,ZHENG Zi-Rui1,ZHANG Yan-Hong1,ZHONG Hui1 1.Beijing Institute of Biotechnology,Beijing 100850,2.Beijing Public Security Hospital,Beijing 100121,3.College of Life Science,Anhui University,Hefei 230039,4.Jinlin University Key Laboratory for Molecular Enzymology , Engineering,Changchun 130012,China |
| Abstract: | Objective:To construct the small interference RNA(siRNA) eukaryotic expressing plasmid of human TBK1(TANK binding kinase-1) and select the cell lines of stable interference TBK1.Methods:According to the reported siRNA sequence and the adhesive end of vector,two of specific DNA sequences were designed and synthesized.After been annealed to generate adhesive end,the oligos were cloned into pSUPER.retro.neo+gfp vector and psiTBK1 was generated.psiTBK1 was transfected into MCF-7 cells by vigorous transfectants.... |
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