血清4型禽腺病毒Ⅷ蛋白真核表达及其对LMH细胞天然免疫相关基因的影响  

Eukaryotic expression of fowl adenovirus serotype 4 protein Ⅷ and its effects on innate immune factors of LMH cell

作　　者：李小凤 阮志华 石勇丽 武晓倩 韦悠 万丽军 谢志勤[1] 任红玉 谢芝勋[1] LI Xiao-feng;RUAN Zhi-hua;SHI Yong-li;WU Xiao-qian;WEI You;WAN Li-jun;XIE Zhi-qin;REN Hong-yu;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,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4期861-867,共7页Southwest China Journal of Agricultural Sciences

基　　金：广西青年科学基金项目(2020GXNSFBA297104);“广西八桂学者”专项(2019A50);广西科技计划项目(AD17195083)。

摘　　要：【目的】分析血清4型禽腺病毒(FAdV-4)Ⅷ蛋白(ProteinⅧ,pⅧ)对宿主天然免疫相关基因表达的影响,为进一步研究FAdV-4的致病机理提供参考依据。【方法】通过PCR扩增FAdV-4Ⅷ基因编码区序列,与pEF1α-HA载体连接,构建重组表达质粒pEF1α-HA-Ⅷ。用pEF1α-HA-Ⅷ重组表达质粒(试验组)和pEF1α-HA空质粒(对照组)转染LMH细胞,利用Western blotting和间接免疫荧光验证Ⅷ蛋白表达情况,实时荧光定量PCR测定STING、MDA5、TBK1、MAVS、NF-κB、IRF7、IFN-α、INF-β、IL-6、IL-8、IL-15和IL-1β基因的表达水平。【结果】Ⅷ基因开放阅读框(ORF)全长744 bp,共编码247个氨基酸残基,真核表达获得的Ⅷ重组蛋白与鼠源HA标签单克隆抗体产生特异性反应,在34 kD处得到单一条带;间接免疫荧光检测可见绿色荧光,说明Ⅷ重组蛋白在LMH细胞中得到良好表达。与对照组相比,Ⅷ重组蛋白在LMH细胞中表达后,模式受体STING基因的表达水平显著提高5.6倍(P<0.05,下同),MDA5基因的表达水平提高30%;接头蛋白TBK1和MAVS基因表达水平分别较对照组显著提高1.5和2.4倍;转录因子IRF7的表达水平显著提高3.4倍,而NF-κB基因被抑制表达。同时,干扰素(IFN-α和IFN-β)和白介素(IL-6、IL-8、IL-15和IL-1β)的表达水平均上调,IFN-α和IFN-β分别极显著上调19.2和14.4倍(P<0.01),而IL-6、IL-8、IL-15和IL-1β分别显著上调4.1、2.1、6.0和7.9倍。【结论】在LMH细胞中过表达FAdV-4 pⅧ,STING、MDA5、TBK1、MAVS、IRF7、IFN-α、IFN-β、IL-6、IL-8、IL-15和IL-1β等天然免疫相关基因呈上调表达,表明Ⅷ蛋白对FAdV-4激发宿主的天然免疫具有一定促进作用。【Objective】The study aimed to explore the influence of fowl adenovirus serotype 4(FAdV-4)proteinⅧ(pⅧ)on expression innate immune-associted genes of host,providing a reference for further study of the pathogenesis of FAdV-4.【Method】FAdV-4Ⅷgene coding sequence(CDS)was amplified by PCR,linked to pEF1α-HA eukaryotic expression vector,and recombinant expression plasmid pEF1α-HA-Ⅷwas 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 proteinⅧwas 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 ofⅧgene was 744 bp,encoding 247 amino acid residues,recombinant proteinⅧcan 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 proteinⅧwas well expressed in LMH cells.Compared with the control group,after the recombinant proteinⅧwas expressed in LMH cells,the expression level of the mode receptor STING gene was significantly increased by 5.6 times(P0.05,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-αand IFN-β)and interleukin(IL-6,IL-8,IL-15 and IL-1β)were up-regulated,and IFN-αand IFN-βwere extremely significantly up-regulated 19.2 and 14.4 times(P0.01),respectively.IL-6,IL-8,IL-15 and IL-1βwere significantly up-regulated 4.1,2.1,6.0 and 7.9 times,respectively.【Conclusion

关 键 词：FAdV-4 Ⅷ蛋白 真核表达 天然免疫因子 实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]