TRAF1-DsRed真核表达载体的构建及其在COS-7细胞中的定位

目的 构建融合蛋白TRAF1-DsRed的真核表达载体及观察TRAF1在COS-7细胞中的表达及定位.方法采用RT-PCR技术从外周血单个核细胞中扩增TRAF1cDNA,定向克隆至真核表达载体pDsRed C1,经脂质体转染COS-7细胞并以间接免疫荧光检测其在COS-7中表达.运用罗丹明123染色和激光共聚焦技术观察TRAF-1在COS-7细胞中的定位.结果转染重组子pTRAF1-DsRed的COS-7细胞可见红色荧光蛋白的表达,TRAF1在COS-7细胞中定位于线粒体.结论 成功构建pTRAF1-DsRed融合基因并进行真核表达,确定TRAF-1在COS-7细胞中的定位,为进一步研究有关TRAF1的信号传导通路奠定了基础.

Construction of recombinant human TRAF1-DsRed fusion protein and its expression and localization in COS-7 cell

Objective To construct a gene expressing vector of fusion protein TRAF1-DsRed and confirm its expression and localization in transfected cell. Methods The TRAF1 target gene was inserted into the pDsRed-C1 vector and then transfected into the COS-7 cell. Local- ization of TRAF1 in the transfected COS-7 cell was detected by immtmohistochemical method, rhodamine 123 staining, and confocol microscopy. Results DsRed was expressed in the COS-7 cell transfected with pTRAF1-DsRed. The TRAF1 protein and the red fluorescence protein were all expressed in the cell. Through confocol microscope, TRAE1 was found localized in mitochondriums. Conclusion pTRAF1-DsRed fusion gene was constructed and TRAF1 was expressed in the transfected COS-7 cells successfully. Data confirm the relationship between over-expression of TRAF1 and the aggregation of mitochondritums.