[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.West-em-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60.0%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FAdV4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.
[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 pEF1α-HA vector,and the re-combinant expression plasmid pEF1α-HA-X was constructed.The recombinant expression plasmid pEF1α-HA-X and empty vector pEF1α-HA-were transfected into LMH cells respectively,after 24 hours,the transfected cells were stimulated with FAdV-4,and the treatments were marked as pEF1α-HA-X+and pEF1α-HA+,without virus stimulation after transfection was set up as control and marked as pEF1α-HA-X-and pEF1α-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(chTLR1a,chTLR1b,chTLR2a,chTLR2b,chTLR3,chTLR4,chTLR5,chTLR7,chTLR15,chTLR21)and effect factors(IFN-α,INF-β,IL-1β,IL-6,IL-8,IL-15).[Re-sult]The open reading frame(ORF)of X gene was 540 bp,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 im-munofluorescence.Real-time florescence quantitative PCR showed that,compared with pEF1α-HA-group the mRNA transcription levels of chTLR1a and chTLR1b in pEF1α-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 pEF1 α-HA-group,mRNA transcription levels of chTLR2a,chTLR2b,chTLR5,chTLR7,chTLR15 and chTLR21 were signifi-cantly up-regulated(P<0.05,the same as below).After virus infection,the mRNA transcription levels of chTLR1a and chTLR1b in pEF1α-HA-X+group were extremely significantly down-regulated by 60.0%and 66.7%compared with pEF1 α-HA-X-group,and the mR-NA transcription levels of chTLR2a,chTLR2b and chTLR3 were significantly increased by 2.91,2.01 and 1.47 times respectively,the tran-scription 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 pEF1α-HA-X+group were significantly up-regulated compared with pEF1α-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,chTLR3)and effector factors(IFN-α,INF-β,IL-1β)significantly up-regulate to inhibit viral replication,indicating that pX has the function of activating the nat-ural immune system of host cells.