南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (08): 1101-1105.

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用FRET技术研究TLR4和MD-2的相互作用

刘亚伟； 刘靖华； 唐靖； 赵清； 李建军； 赵明哲； 李志杰； 王国军； 钟田雨； 邓鹏； 姜勇；   

  1. 南方医科大学基础医学院广东省功能蛋白质组学重点实验室； 南方医科大学基础医学院广东省功能蛋白质组学重点实验室 广东广州510515； 广东广州510515；
  • 出版日期:2006-08-20 发布日期:2006-08-20
  • 基金资助:
    国家973计划研究项目(2002CB513005);国家自然科学基金(30270538)~~

Analysis of Toll-like receptor 4 and myeloid differentiation protein-2 interaction with fluorescence resonance energy transfer

LIU Ya-wei, LIU Jing-hua, TANG Jing, ZHAO Qing, LI Jian-jun, ZHAO Ming-zhe, LI Zhi-jie, WANG Guo-jun, ZHONG Tian-yu, DENG Peng, JIANG Yong Key Laboratory for Functional Proteomics of Guangdong Province, College of Basic Medicine, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学基础医学院广东省功能蛋白质组学重点实验室； 南方医科大学基础医学院广东省功能蛋白质组学重点实验室 广东广州510515； 广东广州510515；
  • Online:2006-08-20 Published:2006-08-20

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摘要： 目的利用荧光共振能量转移(FRET)技术在活体细胞研究人Toll样受体(TLR)4与髓样细胞分化蛋白2(MD-2)的相互作用。方法用PCR方法扩增TLR4编码序列和MD-2编码序列(不包括信号肽序列)并分别亚克隆入带TLR4信号肽的增强型青色荧光蛋白和增强型黄色荧光蛋白表达载体(pECFP-C1-SP,pEYFP-C1-SP)中,重组质粒经酶切、序列鉴定分析后分别或共同转染HEK293细胞,用荧光显微镜观察荧光蛋白表达和分布情况,并用常规的方法和受体光漂白的方法对共表达青色荧光蛋白(CFP)-TLR4和黄色荧光蛋白(YFP)-MD-2的细胞进行FRET分析。结果重组质粒在HEK293细胞中得到表达,仅转染pECFP/TLR4或pEYFP/MD-2的细胞可见青色或黄色荧光主要分布在胞质内,以核周较多;而共转染pECFP/TLR4和pEYFP/MD-2的细胞可同时检测到青色和黄色荧光,主要分布在细胞膜,同时胞质也有少量表达。无论是用常规的方法还是受体光漂白的方法,对共表达CFP-TLR4和YFP-MD-2的细胞进行FRET分析结果表明有FRET现象的发生,提示TLR4和MD-2有相互作用并形成复合物。结论本研究为TLR4和MD-2在活体细胞中的相互作用提供了直接的证据。

Abstract: Objective To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology. Methods The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method. Results The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2. Conclusion This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells. 

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