Duck Toll-Iike Receptor5(TLR5) and Antiviral Molecu es Involve in Anti-Newcastle Disease Virus (NDV) Response
TLR5 is responsible for the recognition of bacterial flagellin in vertebrates.In the present study,the first TLR5 gene in duck was cloned.The ORF of duck TLR5 (dTLR5) cDNA is 2 580 bp and encodes a polypeptide of 859 amino acids.Multiple sequence alignment showed that the amino acid sequence of the dTLR5 is 87.4%,81.3%,50.6% identical to that of goose,chicken and human,respectively.Prediction of protein domains by the SMART program revealed that the putative amino acid sequence consisted of a signal peptide sequence encompassing the first 21 amino acid residues of the N-terminal region,11 leucine-rich repeat (LRR) domains,a leucine-rich repeat C-terminal (LRR-CT) domain a transmembrane domain and a Toll-interleukin-I receptor (TIR) domain at positions 693-840 of the carboxy-terminus.We also cloned partial sequences of TLR5 adaptor protein,myeloid differentiation factor 88 and (MyD88) and the antiviral genes,the gene encoding OAS-like protein (OAS) and Mx in ducks.All these nucleotide sequences were deposited to GenBank.Tissue and cell distribution study showed that the dTLR5 mRNA was highly expressed in the bursa of Fabricius,spleen,trachea.lung,jejunum rectum,skin; moderately expressed in the muscular,glandular tissue,duodenum,ileum,caecum,pancreas; minimally expressed in the heart,liver,kidney,muscle.To study the functionality of dTLR5,this gene was cloned into the pCAGGS expression vector and transfected into the DF-1 and HeLa cell line,along with a NF-KB or IL-6 promoter uciferase reporter plasmid.After flagellin stimulation,the luciferase activities of NF-κB and IL-6 promoter of TLR5-transfected cells were activated significantly when compared to that transfected with empty vector cells.To investigate the effect of NDV on the induction of host immune responses,the expression of dTLR5 mRNA levels in the liver,lung and spleen were detected by qRT-PCR.dTLR5 mRNA expression in liver was significantly increased (2.7-fold,p<0.05) compared to the control at I day post-injection; this expression began to drop (1.7-fold) at 2 day.In lung,TLR5 expression was down-regulated significantly during aLI the test periods (0.37-fold,p<0.05and 0.21-fold,p<0.05).For spleen tissue,the level of TLR5 mRNA was slightly elevated (1.49-fold,p>0.5) at ld.p.i.but this level was not statistically significant when compared to that of the control; at 3d.p.i.this gene was significantly up-regulated (6.65-fold,We analyzed the antiviral genes protein kinase R (PKR) the gene encoding OAS-like protein (OAS) (Figure 6B) and Mx (Figure 6C) known to be IFN-stimulated genes in mouse fibroblasts during infection with Herts33 NDV At ld.p.i,the expression of PKR was significantly up-regulated in both liver (3.6-fold,p<0.05) and spleen (2.7-fold,p<0.05); in lung PKR was slightly elevated (1.99-fold,p> 0.5),but this level was not statistically significant.At 2d.p.i,the PKR was significantly up-regulated in all the tissues tested.especially significantly in spleen (27.6-fold,p<0.05).As for the Mx gene,a similar up -regulation as PKR was found.All the tested tissues except the lung at ld.p.i were significantly up- regulated.This gene was most significantly up-regulated in spleen (20.44-fold,p<0.05).The mRNA expression of OAS gcne was also increased in liver,lung and spleen after Herts33 infection.Unlike Mx and PKR gene,the expression of OAS was especially strong in livcr after infccted with Herts33,while PKR and Mx were stronger in spleen.
duck innate immune response MyD88 NDV TLR5
程玉强 孙英杰 王恒安 严亚贤 李靖 丁铲 孙建和
上海交通大学农业与生物学院 中国农业利学院 上海兽医研究所 山东畜牧兽医职业学院
国内会议
第五届中国兽药大会(中国畜牧兽医学会动物药品学分会2014年学术年会、中国畜牧兽医学会生物制药学分会暨中国微生物学会兽医微生物学专业委员会联合学术论坛)
沈阳
英文
137-149
2014-09-13（万方平台首次上网日期，不代表论文的发表时间）