| The disease of Yersinia Ruckeri(Y.Ruckeri)is a serious acute or chronic bacterial disease of cultured fish caused by Y.Ruckeri.The United States reported in 1999 for the first time that the bacteria were infected with channel catfish and caused serious death.As the athletic organ of Y.Ruckeri,the flagellum is closely related to the pathogenicity and others of the bacterium.Flagellum is mainly composed of flagellin monomer,so the flagellin research is particularly important.The experiment was based on the results of the whole genome sequencing of Y.ruckeri strain SC09,to obtain the gene of FlaA,FlaB and FlaC,and then the molecular characteristics of FlaA,FlaB and FlaC genes were analyzed by bioinformatics software.Subsequently,the three recombinant flagellin proteins were expressed in prokaryotic cells and the specificity of the three expression products was verified by immunoblotting.The immune regulation of three recombinant flagellin were studied in vivo and in vitro.The results are as follows:1 Cloning,expression and bioinformatics analysis of threerecombinant flagellin proteins of Y.ruckeriThree flagellin gene sequences were amplified with genomic DNA of Y.ruckeri used as template.Three sequences of flagellin were analyzed by bioinformatics software.The complete ORF of FlaA,FlaB and FlaC were 1284 bp,1278 bp and 1290 bp,respectively,and the amino acids of the protein were 428,426 and 430,respectively,and the predicted protein molecular mass were 43.5268 KDa,43.63197 KDa and 44.04248 KDa,respectively.The similarity of the three flagellin genes was 72.46%-89.84%,which was medium similar to that of other flagellin amino acid sequences.Domain analysis revealed that all the three flagellin proteins contained a conserved domain of Flagellin_N/C superfamily at the N-terminus and the C-terminus,respectively.And all the three flagellin proteins did not contain signal peptide cleavage sites and transmembrane regions.The results of the phylogenetic tree showed that all the Yersinia species were isolated from a single branch.The three flagellins involved in this paper showed the close homology,and also the closest homology to the reported Yersinia flagellins(Y.Ruckeri(AGL46983)).The purified rF1aA,rFlaB and rFlaC obtained by prokaryotic expression were analyzed and their specificity was also analyzed.The results showed that the optimal expression conditions were as follows:induction temperature 34?,IPTG concentration 0.1 mM,induction time 4 h.And the expression of recombinant protein rFlaA,rFlaB and rFlaC were about 64 KDa.The results of the western-blotting showed that rFlaA,rFlaB and rFlaC had good specificity.2 Comparative study on the immunostimulation of three recombinant flagellin of Y.ruckeriThe three recombinant flagellin cells were used to stimulate the channel catfish cultured head kidney monocytes/macrophages for 4 h,8 h,12 h and 24 h,respectively.The RNA was extracted from each cell at each time point.The expression of TLR5M,TLR5S,NF-?B,MHC??,IL1-?1,TNF?,IL 8,iNOS 1 and Hepcidin was detected by qPCR.The results showed that the three recombinant flagellin proteins could stimulate the increase of the expression of the nine genes.The expression of rFlaC stimulated the above nine genes was higher than that of rFlaA and rFlaB for the above nine gene expression levels.Additionally,the three recombinant flagellin proteins were immunized with healthy and cultured catfish for 8 h,1 d,3 d,1 w,2 w and 4 w,respectively.The RNA was extracted from the head kidney tissue at each time point,and The expression of TLR5M,TLR5S,NF-?B,MHC ??,IL1-?1,TNF a,IL 8,iNOS 1 and Hepcidin was detected by qPCR.The results showed that the three recombinant flagellin proteins could also stimulate the expression of the nine genes,and the three recombinant flagellin found that rFlaC also stimulated the above nine gene expression levels higher than rFlaA and rFlaB for the above nine gene expression. |
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