The Construction Of CD8α + Antigen Cloning Vector And Expression In Dendritic Cells

Objective: To investigate the role of expressed CD8α+ in the immune response of islet cells in the pathogenesis of type 1 diabetes. Molecular cloning of CD8α+ from thymus cells of the NOD mouse, and recombined with lentiviral vector, constructed the expression plasmid encoding CD8α+ and analyzed the sepuences, then cotransfected HEK293 cells by lipofection, obtained virus and re-infected dendritic cells and expressed in it, detected the expression of CD8α+ by Western Blot. Methods: Design the primers of CD8α+ with the CD8α+ sequence.Total RNA was extracted from the thymus lymphocytes in NOD mouse,then was amplified by RT-PCR and recombined with pCDF1-MCS2-EF1-COPGFP plasmid vectors, after DNA sequencing and analyzing, then cotransfected HEK293 cells with pPACKH1TM Lentivector, when packaged recombinant virus, infected DC cells and harvested virus solution, and the expression of CD8α+ was detected by Western Blot. Results: The CD8α+ was successfully cloned with 750bp, when successfully constructed recombinant plasmid between target gene and pCDF1-MCS2-EF1-COPGFP, contrasted with the gene bank, the homology was about 100%. The recombinant plasmid of pCDF1-MCS2-EF1-COPGFP was packaged in HEK293 cells,when cultured for 24 hours, Green fluorescence protein was observed by fluorescence microscopy, obtained virus to infect DC cells,the green fluorescence protein was also seen under fluorescence microscope. And western blot analysis indicated the expression product of CD8α+. Conclusions: After the DNA sequence analysis and immunofluorescence and immunoblotting analysis, the CD8α+ was successfully cloned to the vector,and provided research foundtion for the futhur study of immune mechnisms in type diabetes.