Abstract:

To construct the  recombinant lentivirus vector of CD8α⁺ antigen gene  and to discuss its protein expression in dendritic cells(DCs),and to clarify  the pathogenesis of type 1 diabetes mellitus(T1DM).Methods The specific primer was designed and the full-length gene of CD8α⁺ antigen gene amplified from NOD mice CD8α⁺T lymphocytes were detected by RT-PCR.After plasmid transformation,clone selection,identification and DNA sequencing,the CD8α+ antigen gene were cloned into pCDF1-MCS2-EF1-COPGFP plasmid.The expression of CD8α⁺ antigen gene was analyzed by Western blotting method.The plasmid containing target gene was transfected into HEK293 cells with Nanofectin liposomes transfection reagent for restructuring virus carrier packing.After the DCs were transfected by  the recombinant lentivirus carrier,the expression of  CD8α⁺ protein was  detected by Western blotting method.Results The  recombinant plasmid vector of CD8α+ antigen gene of NOD mice was constructed successfully.After the HEK293 cells were transfected,the stock solution of recombinant  virus  was gotten.After the amplification of recombinant virus,the DCs were infected and the green fluorescent protein (GFP) was  found in DCs under fluorescence microscope by immunofluorescence assay.Conclusion The recombinant virus carrier of CD8α⁺ antigen gene of  NOD mice  is constructed successfully and  the  related proteins can express in it.
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Key words:  T1DM；CD8α⁺ antigen gene；lentiviral vector；gene cloning；dendritic cells