Clone and expression of human cytomegalovirus UL144 gene and its effects on dendritic cells / 第二军医大学学报

ABSTRACT

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.