Expression And Functional Identification Of Recombinant Adenovirus Mediated ShRNA Silencing Of TRAM Gene In Macaca Mulatta Immature Dendritic Cells

[Object] To constructmacaca mulatta TRAM-shRNA adenovirus vector and investigate the inhibiting effect of TRAM-shRNA on immature dendritic cell (imDC) TRAM gene.[Methods]1. PCR identification and sequencing of positive clone after construction of TRAM-shRNA recombination interfering plasmid;2. Packaging of TRAM-shRNA adenovirus vector with HEK293 cells followed by purification of virus using Adeno-XTM purification kit, and quantify the virus titer by endpoint dilution assay;3. Isolation of macaca mulatta PBMC with monkey ficoll-hypaque solution after the collection of macaca mulatta marrow blood;4. Culturing and inducing the PBMC into imDC with IL-4 and GM-CSF;5. Infecting the macaca mulatta imDC with recombinant adenovirus vector, extracting total protein from cells with high GFP expression and quantifying the concentration of protein with BCA assay;6. Investigating the shRNAinhibiting effect on TRAM gene in imDC by applying Western Blot.[Results]PCR amplification and electrophoresis shows the PCR product length of positive clone match the expecting result. Sequencing results demonstrate the accordance of macaca mulatta TRAM-shRNA sequence in positive plasmid with the early synthesized TRAM-shRNA,which further confirmthat the PCR product is the target TRAM-shRNA and indicate the success of recombinant interfering plasmid construction. Quantified the titer of recombinant adenovirus as 2.5×1010PFU/ml after packaging in HEK293 cells and followed purification. Total protein is extracted 48 hours post imDC are transfected with recombinant adenovirus, Western bloting display 32kD target band and 43kD internal reference of P-actin, Gel analysis shows the expression of TRAM protein in TRAM-shRNA interfering group is 98.08% lower than the negative control group, and is 97.73% lower than the blank control group.[Conclusion]The macaca mulatta TRAM-shRNA adenovirus vector has been constructed, and TRAM-shRNA can enter macaca mulatta imDC and inhibit the expression of TRAM protein.