Résumé
Objective: To construct a recombinant adenovirus vector carrying human cytomegalovirus (hCMV) UL144 gene and to explore the biological characteristics of UL144 gene-modified DCs. Methods: The UL144 gene was amplified from hCMV DNA, which was extracted from hCMV-DNA positive serum. The recombinant adenovirus vector carrying hCMV UL144 gene was constructed with AdEasy system and then transfected into HEK293 cells to create recombinant adenovirus Ad-UL144. The expression of inserted gene was identified by RT-PCR. The recombinant adenovirus was then transfected into mice myeloid dendritic cells. The surface proteins of dendritic cells were analyzed by FACS, and cytokines in supernatant were detected by ELISA. T cell proliferation stimulated by gene-modified DC was examined by 3H-TdR uptake assay. Results: The UL144 gene was successfully cloned into the pAdEasy-1 plasmid. The recombinant adenovirus Ad-UL144 was packed in HEK293 cells, with a viral titer of 3×10 10 pfu/ml. DCs infected with AdCMV-UL144 had markedly decreased surface expression of CD80, CD86 and I-Ad (P < 0.01). The contents of TNF-α, IL-6 and IL-1β were significantly decreased in the supernatant of AdCMV-UL144 modified DCs (P < 0.05). T cell proliferation ability induced by gene-modified DC was obviously lower than in the DC control group (P < 0.01). Conclusion: UL144-modified DCs can maintain a relative immature status, and have reduced stimulating activity upon the proliferation and activation of T cells in vitro.
Résumé
Objective:To construct a recombinant adenovirus vector carrying human cytomegalovirus(hCMV) UL144 gene and to explore the biological characteristics of UL144 gene-modified DCs.Methods:The UL144 gene was amplified from hCMV DNA,which was extracted from hCMV-DNA positive serum.The recombinant adenovirus vector carrying hCMV UL144 gene was constructed with AdEasy system and then transfected into HEK293 cells to create recombinant adenovirus Ad-UL144.The expression of inserted gene was identified by RT-PCR.The recombinant adenovirus was then transfected into mice myeloid dendritic cells.The surface proteins of dendritic cells were analyzed by FACS,and cytokines in supernatant were detected by ELISA.T cell proliferation stimulated by gene-modified DC was examined by 3H-TdR uptake assay.Results:The UL144 gene was successfully cloned into the pAdEasy-1 plasmid.The recombinant adenovirus Ad-UL144 was packed in HEK293 cells,with a viral titer of 3?1010 pfu/ml.DCs infected with AdCMV-UL144 had markedly decreased surface expression of CD80,CD86 and I-Ad(P