Expression And Identification Of Recombinant Adenovirus Mediated Macaca Mulatta PD-L1Ig And CD40L Extracellular Region Gene In Macaca Mulatta Immature Dendritic Cells

[Object]To construct and identify the recombinant adenovirus vector with PD-L1Ig and CD40L extracellular region gene of mucaca mulatta. Identify the expression of recombinant adenovirus macaca mulatta PD-L1Ig and CD40L extracellular region gene in macaca mulatta immature dendritic cells.[Methods]1. The construction, identification and packaging of the recombinant adenovirus vector with PD-L1Ig and CD40L extracellular region gene of mucaca mulatta.Inquire the mucaca mulatta PD-L1 and IgG1Fc gene sequence in Gene Bank and composite the gene synthesis of target gene PD-L1Ig, and then amplify the PD-L1Ig with PCR. Concentrate the enzyme-digested product of PD-Lllg gene with the linearized GV282 vector, and then transform the competence E.coli, filtrate transformed bacterial colony with Amp, identify the positive transformant with PCR to get the recombinant plasmid vector:pCMV-3FLAG-IRES-EGFP-PD-LlIg, and then sequencing the vector. Concentrate the enzyme-digested product of pGEM-T-efCD40L plasmid which had been PCR amplified with the linearized recombinant plasmid vector pCMV-3FLAG-IRES-EGFP-PD-L1Ig. And then transform the competence E.coli, filtrate transformed bacterial colony with Amp, identify the positive transformant with PCR to get the recombinant shuttle vector: pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-LlIg and then sequencing the shuttle vector. Transfer 293T cells with the recombinant plasmid vector and then use Western Blot to identify the expression of interest protein. Apply the adenovirus AdMax packaging method to package the recombinant vector and HEK293 cell to get the recombinant adenovirus. Test the titer of the adenovirus by using the end-point dilution.2. Extract and culture macaca mulatta imDC, use the recombinant adenovirus to transfer imDC, apply Western Blot to identify the expression of PD-L1Ig and CD40L extracellular region gene in macaca mulatta imDC.Extract the mononuclear cells in macaca mulatta’s bone marrow blood with the use of monkey lymphocyte separation medium. Using of the magnetic activated cell sorting to extract the CD34+ positive cell in the mononuclear cell. The CD34+ positive cell is induced and differentiated into imDC by the cytokines GM-CSF and IL-4. Confirm the optimum MOI of imDC transfected by recombinant adenovirus. Extracting the protein after 48 hours of the transfection and applying Western Blot respectively detect the protein expression of PD-L1Ig and CD40L extracellular region gene in imDC.[Result]The sequence result of recombinant plasmid vector pCMV-3FLAG-IRES-EGFP-PD-L1Ig is proper. The sequence result of recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig is proper. The recombinant shuttle vector transfer 293T cells, applying Western Blot to test the interest protein, get the specificity stripe in 23kD. The titer of the recombinant adenovirus is 1x10-11 PFU/ml. The recombinant adenovirus transfect macaca mulatta imDC, applying Western Blot to test PD-L1Ig get the specificity stripe in 33kD and Western Blot to test CD40L extracellular region get the specificity stripe in 29kD.[Conclution]The recombinant plasmid vector pCMV-3FLAG-IRES-EGFP-PD-L1Ig was successfully cloned. The recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-Lllg was constructed successfully. Applying the adenovirus AdMax packaging method, we successfully obtain high titer of the recombinant adenovirus. macaca mulatta PD-L1Ig and CD40L extracellular gene can be expressed in macaca mulatta imDC stability.[Prospect]The recombinant adenovirus transfer macaca mulatta imDC successfully and express stability in imDC, and those results can provide theories and experimental foundations to explore the PD-L1Ig and CD40L extracellular region gene inducing the immune tolerance during the vivo experiment of macaca mulatta liver transplantation.