Research On New Methods For Labeling MHC Class I Specifically

Objective: Establish new methods for tracing MHC class I by using site-specific fluorescent protein, and comparing advantages and disadvantages of different methods for labeling MHC class I in somatic cells and antigen-presenting cells(APCs).Methods: (1) Construct eukaryotic lentiviral expression vectors H-2Kb-EGFPTCtag and H-2Kb-TCtag, and produce lentivirus of H-2Kb-EGFPTCtag and H-2Kb-TCtag by transfecting 293FT cells with vectors of H-2Kb-EGFPTCtag and H-2Kb-TCtag, and then infect 293FT cells and DC2.4 cells respectively. At last, label infected 293FT cells and DC2.4 cells with ligands of ReAsH and FlAsH, and observe distribution of H-2Kb with confocal microscope. (2) 293FT cells transfected with vector of H-2Kb-TCtag, and label transfected 293FT cells with ligand of ReAsH, and then observe distribution of H-2Kb with confocal microscope. (3) Construct eukaryotic lentiviral expression vector H-2Kb-halotag, and produce lentivirus of H-2Kb-halotag by transfecting 293FT cells with vectors of H-2Kb-halotag, and then infect 293FT cells and DC2.4 cells respectively. At last, label infected 293FT cells, DC2.4 cells and DC2.4-FUKY cells with ligand of HaloTag?TMR, and observe distribution of H-2Kb with confocal microscope. (4) Construct eukaryotic expression vector H-2Kb-halotagout, and transfected 293FT cells with H-2Kb-halotagout, then label transfected cells with HaloTag?Alexa488. Observe expression and distruibution of H-2Kb with fluorescent microscope.Results: (1) Observe fusion protein kb-EGFP-TCtag and protein labeled with ReAsH, and fusion protein kb-EYFP-halotag and protein labeled with HaloTag?TMR are colocalized with confocal microscope, which prove that binding between TCtag and ReAsH, kbhalotag and HaloTag ? TMR are specific, and do not affect the distribution and expression of kb. (2) Observe distribution of H-2Kb with confocal microscope, finding TCtag and halotag are specific for labeling MHC class I in non-APC cells. (3) Observe distribution of H-2Kb in APC cell, finding TCtag is not specific for labeling MHC class I, but halotag is specific for labeling MHC class I. (4) Observe expression and distruibution of membraneous H-2Kb with fluorescent microscope. Conclusion: This study has successfully established new methods for labeling MHC class I specifically, and studied their characteristics in the labeling process. Results proved that these two methods are simple, convenient, and do not affect expression and distribution of target protein. But they have their own advantages and disadvantages. TCtag is specific for labeling protein only in non-APC cells, but halotag is specific for labeling protein in APC cells and non-APC cells. In addition, TCtag is much smaller than the halotag, and it more suitable for labeling small molecule. Therefore, it will realize labeling various proteins for studying demand.