Construction of wild-type and mutant ABCB6-DsRed fusion protein expression vectors / 医学研究生学报

Objective The mutation of the ABCB6 gene is involved in a variety of diseases , including dyschromatosis univer-salis hereditaria (DUH).This study aimed to construct the expression vectors for the ABCB 6-DsRed fusion proteins, pDsRed-wt-AB-CB6 and pDsRed-L356P-ABCB6, detect its cellular localization in A375 cells, and thus facilitate future studies on the pathogenesis of ABCB6-related diseases . Methods The recombinant plasmids pDsRed-wt/L356 P-ABCB6 were constructed based on the previously constructed pIRES2-ZsGreen1-ABCB6 vector and then transfected into A 375 cells.At 48 hours after transfection , the expression of AB-CB6 was detected by Western blot and the cellular localization of ABCB 6 determined under the laser scanning confocal microscope . Results The expression vectors pDsRed-wt/L356P-ABCB6 were verified by colony PCR, enzyme digestion, and DNA sequencing. The expression of ABCB 6 was significantly increased in the A 375 cells after transfected with the recombinant plasmids .Confocal mi-croscopy showed the localization of both wild-type and mutant ABCB6 in the cytoplasm. Conclusion The expression vectors for wild-type and mutant ABCB6-DsRed fusion protein were successfully constructed and the localization of ABCB 6 in A375 cells was de-termined, which may serve as a basis for further studies of ABCB 6 and the pathogenesis of ABCB 6-related diseases .

Immunotolerance-inducing effect on allogeneic T cells by HLA-G1 expressed on ECV304 / 中华器官移植杂志

Objective To study the inhibitory effects of HLA-G1 expressed in ECV304 on the proliferation of allogeneic T cells.Methods The recombinant plasmid pcDNA3-HLA-G1 which contained a full-length cDNA of HLA-G1 was constructed, and the ECV304 cell was transfected with the pcDNA3-HLA-G1 by using the lipofectin transfection. The expressed HLA-G1 on the cell surface was checked by specific monoclonal antibody (G11E5) with indirect immunofluorescence assay and FCM. The HLA-G1 expressed in ECV304 was used as stimulator in co-culture with allogeneic T cells, to perform allogeneic T cell proliferation assay and to generate ECV304-specific alloreactive CTL. The proliferation of T cells and the CTL’s cytotoxicity against ECV304 were tested by the MTT method.Results The expression of HLA-G1 on the surface of the ECV304 was verified with the immunofluorescent staining of the pcDNA3-HLA-G1 transfected cells. The proliferation intensity of allogeneic T cells was significantly decreased after the HLA-G1 expressed in ECV304, as the stimulation index of co-culture of allogeneic T cells with plasmid pcDNA3 transfected ECV304 was 1.59?0.41, meanwhile it was 1.33?0.46 to pcDNA3-HLA-G1 transfected ECV304, with the difference being significant (P