X 蛋白 (pX) 在血清4型禽腺病毒感染 LMH 细胞后对 Toll 样受体的影响.

[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]

【目的】研究X蛋白 (Protein X, pX) 在血清4型禽腺病毒 (Fowl adenovirus serotype 4, FAdV-4) 感染 LMH 细胞后对 Toll 样受体产生的影响, 为进一步阐释 FAdV-4 感染致病机制和免疫应答机理提供数据参考。【方法】通过 PCR 扩增X 基因编码区序列, 与 pEF1α-HA 载体连接, 构建重组表达质粒 pEF1α-HA-X。将重组表达质粒 pEF1α-HA-X 和空质粒 pEF1α-HA 分别转染 LMH 细胞, 24 h 后用 FAdV-4 感染转染后的细胞, 2 个处理组分别标记为 pEF1α-HA-X+和 pEF1α-HA+; 同时设立转染后未加病毒刺激组作对照, 分别标记为 pEF1α-HA-X 和 pEF1α-HA。用 Western-blotting 和间接免疫荧光验证重组蛋白表达情况, 实时荧光定量 PCR 检测Toll样受体 (chTLR1a、chTLR1b、chTLR2a、chTLR2b、chTLR3、chTLR4、chTLR5、chTLR7、chTLR15、chTLR21) 和效应因子 (IFN-α、IFN-β、IL-1β、IL-6、IL-8、IL-15) mRNA 转录水平的变化。【结果】X基因开放阅读框 (ORF) 全长 540 bp, 共编码 179 个氨基酸残基。Western-blotting 结果显示重组X蛋白与 HA 标签小鼠源单克隆抗体反应, 在 27 kD 处得到单一条带, 间接免疫荧光结果可见绿色荧光。实时荧光定量 PCR 检测发现, 与 pEF1α-HA 组相比较, pEF1α-HA-X 组 chTLR1a 和 chTLR1b 的 mRNA 转录水平极显著上调 (P <0.01, 下同), 分别为 pEF1α-HA 组的 9.76 和 12.34 倍; chTLR2a、chTLR2b、chTLR5、chTLR7、chTLR15 和 chTLR21 的 mRNA 转录水平显著上调 (P< 0.05, 下同)。添加病毒感染后, 与pEF1α-HA-X 组相比, pEF1α-HA-X+组 chTLR1a 和 chTLR1b 的 mRNA 转录水平极显著下调, 分别下调6 0.0% 和 66.7%; chTLR2a、chTLR2b 和 chTLR3 的 mRNA 转录水平显著提高, 分别显著上调 2.91、2.01 和 1.47 倍, 其他 chTLRs 的 mRNA 转录水平下调, 但差异不显著 (P>0.05); 同时, pEF1α-HA-X+组效应因子 IFN-α、IFN-β、IL-1β 的 mRNA 转录水平比 pEF1α-HA-X 组显著上调, IL-8 的 mRNA 转录水平极显著下调。【结论】在 LMH 细胞过表达 pX 并被 FAdV-4 感染后, pX 能激活宿主免疫应答系统, Toll 样受体 (chTLR2a、chTLR2b 和 chTLR3) 和其效应因子 (IFN-α、IFN-β和IL-1β) 的 mRNA 转录水平显著上调以抑制病毒复制,说明pX具备激活宿主细胞天然免疫系统的功能。 [ABSTRACT FROM AUTHOR]