Résumé
Objective To construct an eukaryotic expression vector for TAP genes fused with enhanced green fluorescent protein(EGFP)gene,and to analyze the expression and subcellular localization of the fusion protein in A375 human malignant melanoma cells transfected with the eukaryotic expression vector.Methods A375 cells were cotransfected with the combination of plasmid(P)TAP1-EGFP or pTAP2-EGFP and pDsRed2-endoplasmic reticulum(ER),or with pEGFP-TAP1 and-TAP2,or monotransfected with pTAP1-EGFP or pTAP2-EGFP alone.The monoclonal A375 cells stably expressing TAP were obtained by G418 selection.Then.the distribution and expression of fusion proteins were assessed in A375 cells by using fluorescence microscopy and Western blot,respectively.Flow cytometry was used to measure the expression of HLA class Ⅰ on A375 cells.Results Transfection of A375 cells with pTAP1-EGFP or pTAP1-EGFP and pTAP2-EGFP significantly increased the expression of TAP 1 and TAP 2 in as well as HLA class Ⅰ antigen on A375 cells.The green fluorescence of TAP1-EGFP and TAP2-EGFP overlapped with the red fluorescence of ER marker in cotransfected cells.indicating that TAP was localized subcellularly on the ER.Conclusions The expression vector for TAP-EGFP fusion gene has been constructed cuccessfully and expressed in A375 cells,and the expressed fusion protein is subcelluiarly localized to ER.This study will provide a basis for the research into subsequent immune response following induction of TAP expression.