Constructing a recombinant adenovirus carrying human cytomegalovirus UL138 gene and evaluating the effects of UL138 gene on the functions of monocytes
10.3760/cma.j.issn.0254-5101.2016.09.005
- VernacularTitle:HCMV潜伏相关UL138基因重组腺病毒的构建及其对THP-1单核细胞功能的影响
- Author:
Gangqiang GUO
;
Jing CHEN
;
Wenjing CHEN
;
Lulu YE
;
Xiangwei SUN
;
Liang ZHANG
;
Huidi ZHANG
;
Qiao′ai LIN
;
Xian SHEN
;
Lifang ZHANG
;
Xiangyang XUE
- Publication Type:Journal Article
- Keywords:
Human cytomegalovirus;
Latent infection;
UL138;
Recombinant adenovirus;
THP-1 mononuclear cell;
Chemokine and receptor
- From:
Chinese Journal of Microbiology and Immunology
2016;36(9):667-675
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant adenovirus carrying UL138 gene, which was re-lated to the latent infection of human cytomegalovirus, and to investigate the effects of UL138 gene on the functions of THP-1 mononuclear cells. Methods The recombinant adenovirus expressing the UL138 gene was packaged. The titer of the recombinant adenovirus was determined by calculating 50% tissue culture in-fective dose ( TCID50 ) . THP-1 mononuclear cells were infected with the recombinant adenovirus at different multiplicity of infection (MOI) and the optimal MOI was determined (100 PFU/cell) by observing the ex-pression of green fluorescent protein ( GFP ) . Changes in the expression of proinflammatory cytokines by THP-1 mononuclear cells that was induced by overexpressed UL138 were analyzed by quantitative PCR. The expression of chemokines and their receptors were measured by quantitative PCR array. Results The re-combinant adenovirus carrying the UL138 gene was successfully constructed with a titer of 1×1011 PFU/ml. The rate of THP-1 mononuclear cells that was infected with the recombinant adenovirus was 60% at the MOI of 1 ∶ 100. Results of the RT-PCR analysis and Western blot assay further confirmed that the recombinant adenovirus could infect THP-1 mononuclear cells successfully and the expression of UL138 protein increased gradually over time. The overexpressed UL138 in THP-1 mononuclear cells significantly inhibited the expres-sion of IL-18, IL-1β, IL-6, IL-8 and TNF-α as indicated by the results of quantitative PCR. Results ob-tained from the quantitative PCR array analysis showed that most of the chemokines and their receptors were downregulated in the transfected THP-1 mononuclear cells except for the chemokines of CCL17, CCL21, CCL2 and XCL2 and the receptors of CCR2, CXCR1, CXCR2, CXCR4 and CX3CR1 which were upregulat-ed. Conclusion We successfully constructed the recombinant adenovirus carrying UL138 gene which could be used to infect THP-1 mononuclear cells. Overexpressed UL138 in THP-1 mononuclear cells significantly affected the functions of THP-1 mononuclear cells.
