Expression And Identification Of Recombinant Lentivirus Mediated Macaca Mulatta Antisense MiRlet-7i In Rhesus Monkey Immature Dendritic Cells

Objectives:Construction and identification of rhesus monkey antisense miRlet-7i(Micro RNAlet-7i)lentivirus vector;induction and culture of immature dendritic cells in rhesus monkey;transfection of rhesus monkey immature dendritic cells with recombinant antisense miRlet-7i lentiviral vector;PCR(Polymerase Chain Reaction)was used to detect the expression of miRlet-7i in rhesus monkey immature dendritic cells.Methods:1.Lentiviral vector was digested and ligated with the annealed double stranded DNA of the target gene.The product was added into the competent cells.Then sequencing the cultured vector.Packaging lentiviral vector.After concentrated and purified,the titer of the virus was determined using fluorescence method2.Mononuclear cells were isolated from peripheral blood of rhesus monkey was used to induce and culture immature dendritic cells in vitro by adding cytokine GM-CSF(Granulocyte-Macrophage Colony Stimulating Factor)(50ng/ml)and IL-4(Interleukin-4)(50ng/ml).Immunophenotype identification and ultrastructural observation were carried out by flow cytometry and scanning electron microscope.3.rhesus monkey immature dendritic cells were transfected with recombinant lentiviral vector.RT-q PCR(Real-time Quantitative Polymerase Chain Reaction)was used to detect the expression of miRlet-7i in rhesus monkey immature dendritic cells.Results:The result of sequencing showed that the target sequence was consistent with the previous design sequence(AGCGCCGTCTTTTTTCTAG).After cell packing and purification,the titer of the virus was determined to be 1.6E+8TU/ml using fluorescence method.Dendrites and branches were observed in the induced and cultured cells under 200×and 400×light microscope.The results of flow cytometry and scanning electron microscopy showed that the immunophenotype and ultrastructure of the cells were consistent with those of immature dendritic cells.The recombinant lentiviral vector was transfected into rhesus monkey immature dendritic cells.The results of RT-qPCR analysis showed that The inhibition of miRlet-7iConclusions:Construction of rhesus monkey antisense miRlet-7i lentiviral vector by ligating antisense miRlet-7i with lentivirus vector.Mononuclear cells in peripheral blood of rhesus monkey were successfully cultured and induced into immature dendritic cell of Macaca mulatta.Then immature dendritic cells were infected with recombinant lentiviral vector.The down regulation of miRlet-7i?TNF-??TLR4?NF-?Bwas detected by RT-qPCR,and the target gene was successfully expressed in the target cells.Prospect:Transfect the immature dendritic cells of Macaca mulatta with recombinant rhesus monkey antisense miRlet-7i lentiviral vectors to obtain immunologically tolerant dendritic cells.Dendritic cells have laid a good foundation in the study of immune tolerance in rhesus monkey liver transplantation.