The Clone,Expression And Immune Response Against Salmonella Of Dog Toll-like Receptor 5 Gene

The structure of Toll-like receptor is the typical type I transmembrane structure.It worked as a bridge in the nonspecific immunity and specific immune.They activate the downstream immune pathway by monitoring and recognizing different molecular patterns associated with disease.They are not only the doorkeeper of the innate immunity,but also the link of innate immunity and acquired immunity.Toll-like receptor 5 is a member of toll-like receptor family,It had the outstanding trait in pattern recognition receptors,the main ligand recognized by toll-like receptor 5 is bacterial flagella protein.Recent years intestines problem which was raised by salmonella enteritidis kept climbing,the main reason was that pathogenesis of salmonella enteritidis kept unknown.The molecular mechanisms of toll-like receptor 5 in immune response of salmonella enteritidis was preliminarily studied.We wanted to provide more choices for the treatment of dog's intestines problem caused by salmonella enteritidis.We also wanted to provide theoretical foundation for the breeding of good varieties of dogs.The details of our study was as follows:1.The clone and sequence analysis of dog TLR5 geneIn this study,specific primers was designed according to CDS area of the dog's toll-like receptor 5 gene published in Gen Bank.The dog's toll-like receptor 5 gene was obtained by PCR from the dog's genomic DNA,then constructed the recombinant plasmid pCR2.1-DogTLR5,the sequential characterization of dog TLR5 gene was analysed by using bioinformatics softwares after sequencing.The results were as follows:The gene sequence cloned by us shared 98% homology with dog TLR5 sequence(NC 006620)published in Gen Bank.The open reading frame of dog TLR5 gene was 2577 bp,which coded 858 amino acids.Protein molecular mass of dog TLR5 was 94.6757 kDa.The isoelectric point of dog TLR5 was 8.57.Instability index of the dog TLR5 is 43.14,which showed that dog TLR5 protein is Instability.The protein domains of dog TLR5 consisted of a signal peptide sequence in the N-terminal region(the first 21 aminoacid residues).The protein structure of dog TLR5 consist of a extracellular region which had 8 leucine-rich fragments,a transmembrane domain and a intracellular region.The result of homology alignment showed that dog TLR5 had high homology with mammals such as panda and manchurian tiger.Dog TLR5 had 78.7% homology with panda and 76.8% homology with manchurian tiger.Dog TLR5 had low homology with poultry such as chick,dog TLR5 had 52.7% homology with chick.2.Innate immune responses of dog TLR5 lines against infection with the bacterial flagellum proteinOn the premise of the right sequencing of dog TLR5,the eukaryotic expression vector pcDNA3.1(+)-DogTLR5 was structured.Then the pcDNA3.1(+)-DogTLR5 and the reporter vector pGL4.32[luc2P/NF-?B-RE/Hygro] were used to co-transfect to HEK293 T cells.The co-transfected HEK293 T cells were stimulated by flic.The activity of nuclear transcription factor NF-?B was detected.In addition,we detected the secretion of IL-8 protein in cultured cells supernatant by ELISA.The results were as follows:the NF-?B activated highly by dog TLR5 after the stimulation of flic(P<0.001).The supernatant Of cells stimulated by flic had highly significant difference(P<0.001)with blank group in expression quantity of IL-8.This work lays a theoretical foundation for the rearch of interaction between dog TLR5 and flic.3.Innate immune responses of dog PBMCs lines against infection with the bacterial flagellum proteinThe dog PBMCs were infected in vitro by salmonella enteritidis,the expression quantity of mRNA of dog TLR5 and the downstream inflammatory cytokines were detected at 2 h?4 h?8 h and12 h post-infection by the relative fluorescence quantitative.The results were as follows: The expression quantity peaks of IL-8 gene and IL-1?gene were much more than the expression quantity peaks of IL-6 gene,which indicated that dog TLR5 gene could activated increased expression of IL-8 gene and IL-1? gene but it could not activated increased expression of IL-6 gene.