信号肽-FRET荧光蛋白载体的构建及在NIH3T3细胞中的表达

Construction of signal peptide-FRET fusion expression vectors and their expressions in NIH3T3 cells

目的构建带TLR4信号肽的增强型青色荧光蛋白(pECFP-C1-SP)和增强型黄色荧光蛋白(pEYFP-C1-SP)的质粒载体,为使用荧光共振能量转移(FRET)技术研究LPS在细胞膜上的受体识别机制提供依据。方法采用点突变技术构建了带TLR4信号肽的载体,用脂质体瞬时转染NIH3T3细胞,观察带信号肽载体和融合蛋白载体在细胞内的表达。结果DNA测序证明所构建的带TLR4信号肽的载体正确;酶切和DNA测序表明融合蛋白载体的构建正确,TLR4信号肽可以使蛋白主要表达在膜上。结论所构建的带信号肽的荧光蛋白载体是正确的,pECFP-C1-SP和pEYFP-C1-SP能够表达在细胞膜上,可有效地应用于膜蛋白相互作用的研究。

Objective To construct the fluorescence protein vector pairs with toll-like receptor 4 (TLR4) signal peptide, namely pECFP-C1-SP and pEYFP-C1-SP, therefore to make the application of fluorescence resonance energy transfer (FRET) possible in the study of the membrane protein interaction during lipopolysaccharide (LPS) recognition. Methods pECFP-C1-SP and pEYFP-C1-SP were constructed by means of site-directed mutagenesis, and the constructed plasmids were transiently transfected into NIH3T3 cells via lipofectamin to observe their intracellular expressions under a fluorescence microscope. Results DNA sequence analysis attested the validity of the constructed fluorescence vectors with signal peptide for FRET, and the expression of the vectors was located principally on the cell membrane as observed under fluorescence microscope. Conclusion The constructed vectors TLR4 signal peptide are valid and capable of expressing on the cell membrane, therefore they can be effectively used in the study of the interaction between the membrane proteins.