【摘要】 目的　构建GFP与H 2Kb 融合基因 ,并分析融合基因在活细胞中的表达。方法　应用基因工程技术构建H 2Kb 与绿色荧光蛋白 (GFP)的融合基因。RT PCR和Western印迹分析基因表达 ;激光共聚焦荧光显微镜观察活细胞内荧光布局。结果　RT PCR和Western印迹分析表明融合基因获得了正确表达。激光共聚焦荧光显微镜观察结果表明 ,Kb GFP融合分子象天然Kb 分子一样表达在细胞膜表面 ,且胞外区域可被Kb 分子特异性单克隆抗体所识别 ,表明Kb GFP融合分子可在细胞内正确折叠和装配。Kb GFP融合基因的瞬间和稳定表达均获得了相同结果。结论　Kb GFP融合蛋白具有GFP的自发荧光特性 ,且不影响Kb 分子在细胞内的正确表达。更多还原

【Abstract】 Objective To construct a fused gene of H 2K b and green fluorescent protein, and analyze the expression of fused K b GFP in living cells. Methods A fusion gene composed of green fluorescent protein (GFP) and murine class I MHC molecule H 2K b cDNA was constructed by standard cloning technology. Northern blot and Western blot analysis were used to evaluate the expression of the fused gene. Laser scanning confocal microscopy was used to monitor the distribution of green fluorescence. Results Laser scanning confocal microscopy clearly revealed that the chimeric K b GFP molecules were mostly expressed on the membrane surface as the natural counterpart. Furthermore, the extracellular portion of the expressed K b GFP conjugate can be recognized by a monoclonal antibody specific for folded K b molecule, implying the correct folding and assembly of GFP tagged K b molecules. K b GFP can be transiently and stably expressed in mammalian cells. Conclusion The GFP tag does not interfere with the natural assembly and transport of K b molecule, and K b GFP can act as a potential tool to learn more about antigen presentation by class I MHC molecules in living cells.更多还原