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中文摘要：
目的采用位点特异性荧光蛋白标记技术建立新型示踪MHC-I类分子的方法,比较TCtag和halotag在体细胞和抗原递呈细胞中示踪MHC-I类分子的优缺点。方法构建H-2Kb-TCtag和H-2Kb-halotag融合蛋白的真核慢病毒表达载体,转染293FT细胞制备病毒,将病毒分别感染体细胞293FT和树突状细胞系DC2.4细胞后,TCtag采用染料ReAsH和FlAsH染色,Halotag采用染料HaloTagTMR染色,在激光共聚焦显微镜下观察H-2Kb的分布情况。结果通过激光共聚焦显微镜观察发现：TCtag与染料ReAsH和FlAsH只在293FT细胞内是特异性的结合;Halotag与用染料HaloTagTMR在293FT和DC2.4细胞内都是特异性结合的。结论从结合特异性上来看,Halotag标记MHC I类分子要优于TC-tag。
英文摘要：
This study is aimed to establish new methods for tracing MHC class I by using site-specific fluorescent protein and to compare the advantages and disadvantages of Tetracysteine tag （TCtag） and Halotag in labeling MHC class I molecules in somatic cells （239FT） and antigen-presenting cells （DC2.4）. Lentiviral expressing vectors of H-2Kb-TCtag and H-2Kb-Halotag were constructed and transiently transfected into 293FT cells for preparing lentivirus,which were subsequently used to infect 239FT and DC2.4 cells respectively for expressing MHC class I molecules. Then cells with TCtag labeled with ReAsH or FlAsH,while Halotag with HaloTagTMR. Finally the distribution of H2-Kb was observed by confocal microscope. We found that only in 293FT cells,the binding of TCtag with ReAsH or FlAsH were specific,while the binding of halotag with HaloTag TMR was specific in both 293FT and DC2.4 cells. All these results suggested that the binding specificity of Halotag is much better than that of TC-tag for labeling MHC class I molecules.
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