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  

Effect of X protein(pX)on Toll⁃like receptors after LMH cell infection with fowl adenovirus serotype 4

作　　者：李小凤 韦悠 罗思思[1] 谢志勤[1] 阮志华 张民秀[1] 黄娇玲 李丹[1] 万丽军 李孟[1] 任红玉 谢丽基[1] 谢芝勋[1] LI Xiao-feng;WEI You;LUO Si-si;XIE Zhi-qin;RUAN Zhi-hua;ZHANG Min-xiu;HUANG Jiao-ling;LI Dan;WAN Li-jun;LI Meng;REN Hong-yu;XIE Li-ji;XIE Zhi-xun(Guangxi Key Laboratory of Veterinary Biotechnology/Guangxi Veterinary Research Institute/Key Laboratory of China(Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control,Ministry of Agriculture and Rural Affairs of China,Nanning 530001,China)

机构地区：[1]广西兽医生物技术重点实验室/广西兽医研究所/农业农村部中国(广西)—东盟跨境动物疫病防控重点实验室,南宁530001

出　　处：《西南农业学报》2023年第12期2814-2821,共8页Southwest China Journal of Agricultural Sciences

基　　金：广西科技计划项目(AD17195083);广西农业农村厅基本科研业务费专项(桂科专项23-2);广西青年科学基金项目(2020GXNSFBA297104);“广西八桂学者”专项(2019A50);广西兽医生物技术重点实验室自主研究项目(20-065-23-A-1)。

摘　　要：【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-�【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 pEF1α⁃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+grou

关 键 词：FAdV-4 PX TOLL样受体 效应因子 实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]