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【Objective】The study aimed to explore the influence of fowl adenovirus serotype 4(FAdV-4) protein Ⅷ(pⅧ) on expression innate immune-associted genes of host, providing a reference for further study of the pathogenesis of FAdV-4.【Method】FAdV-4Ⅷ gene coding sequence(CDS) was amplified by PCR,linked to pEF1α-HA eukaryotic expression vector, and recombinant expression plasmid pEF1α-HA-Ⅷ was constructed. Recombinant expression plasmid pEF1α-HA-Ⅷ(experimental group) and empty plasmid pEF1α-HA(control group) were transfected into LMH cells, and the expression of protein Ⅷ was verified by Western blotting and indirect immunofluorescence, real-time florescence quantitative PCR was used to detected the expression levels of STING, MDA5, TBK1, MAVS, NF-κB, IRF7, IFN-α, INF-β, IL-6, IL-8, IL-15, IL-1β.【Result】The open reading frame(ORF) length of Ⅷ gene was 744 bp, encoding 247 amino acid residues, recombinant protein Ⅷ can react with HA-tag monoclonal antibody from mouse, a single band was obtained at 34 kD, and green fluorescence was observed by immunofluorescence detection, indicating that recombinant protein Ⅷ was well expressed in LMH cells. Compared with the control group, after the recombinant protein Ⅷ was expressed in LMH cells, the expression level of the mode receptor STING gene was significantly increased by 5.6 times(P＜0.05, the same as below), and the expression level of MDA5 gene was increased by 30%. The gene expression levels of adaptor protein TBK1 and MAVS genes were significantly increased by 1.5 and 2.4 times compared with the control group, respectively. The level of transcription factor IRF7 was significantly increased by 3.4 times, and the expressions of NF-κB genes were suppressed. Meanwhile, the expression levels of interferon(IFN-α and IFN-β) and interleukin(IL-6, IL-8, IL-15 and IL-1β) were up-regulated, and IFN-α and IFN-β were extremely significantly up-regulated 19.2 and 14.4 times(P＜0.01), respectively. IL-6, IL-8, IL-15 and IL-1β were significantly up-regulated 4.1, 2.1, 6.0 and 7.9 times, respectively.【Conclusion】Overexpression of FAdV-4 pⅧ leads to up-regulated expression of STING, MDA5, TBK1, MAVS, IRF7, IFN-α,IFN-β, IL-6, IL-8, IL-15 and IL-1β in LMH cells. The results indicate that protein Ⅷ can promote the natural immunity of host stimulated by FAdV-4 to a certain extent. [ABSTRACT FROM AUTHOR]

[Objective] To study the effects of protein X (pX) on Toll-like receptors after fowl adenovirus serotype 4 (FAdV-4) infection of LMH cells, the present paper provided data reference for further explaining the pathogenic mechanism and immune response mechanism of FAdV-4 infection. [Method] FAdV-4 X gene coding sequence (CDS) was amplified by PCR, and linked to pEFl α-HA vector, and the recombinant expression plasmid pEFlα-HA-X was constructed. The recombinant expression plasmid pEFlα-HA-X and empty vector pEFlα-HA-were transfected into LMH cells respectively, after 24 hours, the transfected cells were stimulated with FAdV-4, and the treatments were marked as pEFlα-HA-X+ and pEFlα-HA+, without virus stimulation after transfection was set up as control and marked as pEFlα-HAX- and pEFlα-HA-. Western-blotting and indirect immunofluorescence were used to verified expression of recombinant protein X, real-time florescence quantitative PCR assays was used to detected the mRNA expression levels of Toll-like receptors (chTLRla, chTLRlb, chTLR2a, chTLR2b, chTLRJ, chTLR4, chTLR5, chTLR7, chTLR15, chTLR21) and effect factors (JFN-α, INF-β, IL-1β, IL-6, IL-8, IL-15). [Result] The open reading frame (ORF) of X gene was 540 hp, encoding 179 amino acids residues. Western-blotting showed that recombinant X protein reacted with HA tag mouse-derived monoclonal antibody with a single band at 27 kD and green fluorescence visualized by indirect immunofluorescence. Real-time florescence quantitative PCR showed that, compared with pEFlα-HA-group the mRNA transcription levels of chTLRla and chTLRl b in pEFl α-HA-X-group were extremely significantly up-regulated (P < 0. 01, the same as below), as 9. 76 and 12. 34 times as that of pEFl α-HA-group, mRNA transcription levels of chTLR2a, chTLR2b, chTLR5, chTLR7, chTLRl 5 and chTLR21 were significantly up-regulated (P < 0. 05, the same as below). After virus infection, the mRNA transcription levels of chTLRl a and chTLRl b in pEFlα-HA-X+ group were extremely significantly down-regulated by 60. 0% and 66. 7% compared with pEFlα-HA-X-group, and the mRNA transcription levels of chTLR2a, chTLR2b and chTLRJ were significantly increased by 2. 91, 2. 01 and 1. 47 times respectively, the transcription levels of other chTLRs were down-regulated but not significantly ( P > 0. 05). At the same time, the mRNA transcription levels of IFN-α, IFN-β, IL-1β in pEFlα-HA-X + group were significantly up-regulated compared with pEFlα-HA-X-group, mRNA transcription level of IL-8 was extremely significantly down-regulated. [Conclusion] Overexpression of pX in LMH cells and after infection with FAdV-4, pX can activate host immune response system, mRNA transcription level of Toll-like receptors (chTLR2a, chTLR2b, chTLRJ) and effector factors (JFN-α, INF-β, IL-1β) significantly up-regulate to inhibit viral replication, indicating that pX has the function of activating the natural immune system of host cells. [ABSTRACT FROM AUTHOR]