新型示踪MHC-I类分子方法的建立

Establishment of new methods for tracing MHC class I molecules

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HaloTagTMR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HaloTagTMR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。

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 HaloTagTMR. 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.