Expression And Identification Of Recombinant Adenovirus Mediated Macaca Mulatta HLA-G Gene In Macaca Mulatta Immature Dendritic Cells

[Object]To construct the HLA-G overexpression adenovirus vector of macaca mulatta and identify it; Macaca mulatta immature dendritic cells (imDC) which were cultured and filtered were transfected with recombinant adenoviral vector, and expression of HLA-G purpose gene is detected by western blot.[Methods](一) Construction of overexpression adenovirus vector:the purpose vector and plasmid containing the target gene was digested, then the digestion products were exchanged after electrophoresis recyling, its products were transformed into competent bacteria cells. Colony PCR identification of the clone of growing, after that the PCR identification of positive clones were sequenced and compared to the analysis, and the comparison results correct was the purpose of building a successful plasmid.(二) Detection of purpose plasmid expression:293T cells were transfected by overexpression eukaryotic plasmid of mucaca mulatta HLA-G target genes, and western blot was applied to detect the expression of the target plasmid.(三) Mucaca mulatta HLA-G overexpression adenoviral vector were packaged and amplified through adMax adenovirus packaging system,in the next moment Adeno-X Purification Kit purified virus and adenoviral titer were measured with end-point dilution.(四)Mucaca mulatta dendritic cells were fostered and adenovirus infection efficiency was detected.(五) The expression of HLA-G in mucaca mulatta immature dendritic cells were detected by western blot.[Result]Colony PCR identification of the clone of growing, positive transformants PCR product size was 618bp,which was consistent with the theoretical value.PCR identification of positive clones were sequenced and comparative analysis, and the comparison result was correct,it indicated that the target plasmid was constructed successfully. Target plasmid transfected HEK293 cells were detected by western blot that could be observed with characteristic bands near 18KD, and its size in conformity with the purpose gene fusion protein.The results showed that the target gene overexpression is OK. Adenoviral titer was 1×1011/ml measured by the end point dilution. Mucaca mulatta immature dendritic cells were infected by recombinant adenovirus,and western blot detection saw 42KD occurring at specific bands that were consistent with the target protein size.[Conclution]Construction of mucaca mulatta HLA-G adenovirus vector by means of connecting HLA-G with adenovirus vector successfully, then adenovirus vector was used to infect mucaca mulatta immature dendritic cells,and HLA-G could be stably expressed in mucaca mulatta imDC after western blot.[Prospect]Immature dendritic cells were infected by recombinant adenovirus, hoping to obtain tolerogenic dendritic cells,and dendritic cells will be injected into the portal vein of receptor to study mucaca mulatta liver transplantation tolerance in the next step.