| Streptococcus iniae?S.iniae?,an important aquatic pathogen,has infections reported in various species worldwide,not only endangering the development of aquaculture industry,but also threatening the global fish diversity.Although the research of the S.iniae vaccine has advanced,the commercial vaccines are still in research and development stage.With liable security,good stability and high production,subunit vaccines are highly appreciated.Therefore,selection of immunoproteins with high conservation and strong immunogenicity are the core of the efficient research and development of subunit vaccines.This study analyzed the whole genome sequence of S.iniae isolated from the channel catfish,discussed the adaptation mechanism of the host and the pathogenesis of S.iniae,by combing with the other 8 reported S.iniae genome sequences.Through bioinformatics analysis,5 conservative vaccine candidate genes with strong immunogenicity were selected and the corresponding proteins with strong immunogenicity and antigenicity were obtained.Results of the protective efficacy trials on channel catfish showed that the immune protective effect of rSrr is the highest among all candidate proteins.1.The whole genome sequencing of Streptococcus iniae DX09The 16S rDNA gene of S.iniae DX09 was identified by using S.iniae DX09 as the PCR template,blasted with other 16S rDNA sequences from common aquatic pathogenic bacteria in the Genbank,and analyzed by using the Neighbor-Joining methods for phylogenetic analysis.Results showed that the 16S rDNA sequence of strain DX09 clustered with other S.iniae 16S rDNA sequences,which indicated that the strain DX09 was a S.iniae sequence.Observation results of the pathogenicity of DX09 showed that the spleen of infected channel catfish turned black and swelling,resulting in obvious hyperemia and hemorrhage in the spleen tissue,and the DX09 strain survive and reproduce in spleen cells.The genome DNA of DX09 was extracted for the Illumina Miseq sequencing.Results showed the sequencing library has uniform coverage and is capable for further analysis.Using SOAPdenovo v2.04 stitching software to multiple Kmer parameters optimization sequence splicing sequence assembly,functional annotation and conservative immunogenicity gene cluster prediction;Using GapCloser v1.12 software to fill the local inner hole of the result of assembling base and to conduct base correction.Additionally,predictive results of the rRNA/tRNA analysis suggested that the DX09 genome contained 1893 genes,with 1719147 bp in length and 37.1%G+C content.A comparative analysis was taken among with the DX09 gene sequence and the sequences of immunoproteins on the S.iniae membrane surface?including a fibronectin binding protein FbpA,a ferric ion carrier Ftp,a neuraminidase NeuA,a repeated serine protein Srr and a predictive secretory protein K710-1065?.Results showed that FbpA,Ftp,Neu,Srr,and K710-1065 were high conservative among the 9 S.iniae strains,which suggested that these 5 proteins can be regarded as candidate proteins for subunit vaccine.2.The comparative genomic research of S.iniae DX09A comparative genomic research was taken among with the S.iniae DX09 with 4 other S.iniae complete genomes?including S.iniae YSFST01-82,S.iniae SF1,S.iniae ISET0901,and S.iniae ISNO?and 4 S.iniae draft genomes?including S.iniae 9117,S.iniae CAIM 527,S.iniae IUSA1,and S.iniae KCTC 11634BP?to analyze the genomic characteristics of S.iniae and the biological adaptability and some unique laws of evolution.The relationship between the DX09 and YSFST01-82 strains with other S.iniae in the plasticity zone I?PZ-1?and plasticity zone II?PZ-II?was analyzed by the BLAST Ring Image Generator?BRIG?.Results showed that PZ-I is about 18 Kb and exists in DX09,ISET0901,ISNO,CCAIM 527,IUSA1 and SF1,but not in YSFST01-8,KCTC 11634 BP and 9117.Analysis results of the PZ-I sequence in DX09 suggested that several genes were associated with the type VII secretion system,a gene cluster composed of 4 genome,a O-glycosylation hydrolase and some putative proteins.Furthermore,assess the missing area in the DX09 strain by applying the S.iniae YSFST01-82 strain,whose genome is complete,as a target and reference genome.Results showed that DX09 lack a 28 kb PZ-II.Sequence analysis indicated that PZ-II has 4 mobile elements,including 3 genes with transposase domain?SI8201055,SI82 01060 and SI82 01065?which were identified as inserted sequences?ISs?,and another gene?SI82 01065?could encode an integrase belonged to the rve family.Results of the genes analysis of the conserved domain in PZ-II indicated that these genes were associated with the phosphoenolpyruvate-carbohydrate phosphotransferase system?PTS?.Above showed that S.iniae had two major plasticity areas,encoding a type VII secretion system and a PTS system respectively,which reflected the adaptability and the molecular nature of the bacteria surviving in the specific host.3.The bioinformatic analysis of the vaccine candidate protein of S.iniae DX09Results of bioinformatics analysis showed that the 5 gene-based sequence were 3585 bp?Snr?,2709 bp?NeuA?,957 bp?K710-1065?,1734 bp?FbpA?and 3759 bp?Ftp?respectively,and the putative proteins were 124.32 kDa?Srr?,101.38kDa?NeuA?,34.18 kDa?K710-1065?,66?20 kDa?FbpA?and 140.35 kDa?Ftp?correspondingly.By Blastp,results showed that these 5 proteins had high consistency with other S.iniae related proteins,and results of the antigenic prediction suggested that they had strong antigenicity,which providing a more adequate theoretical basis for the subsequent vaccine research.4.Prokaryotic expression and immunogenicity analysis of 5 vaccine candidate protein of S.iniae DX09Based on the bioinformatics analysis,construct the recombinant prokaryotic expression plasmid and expression of the 5 candidate proteins,rSrr,rNeuA,rK710,rFbpA and rFe,were induced with IPTG.The SDS-PAGE analysis of protein distribution suggested that after ultrasonication,rSRR and rK710 mainly expressed in the supernatant in the form of intracellular soluble expression but less in sediment,while rNEUA rFbpA and rFtp mainly expressed in sediment in the form of inclusion body,but less or not in the supernatant.Purify 5 recombinant proteins by the Ni-NTA affinity chromatography method,and analyze their immunogenicity in vitro by Western Blot.Results showed that specific response were occurred between these 5 recombinant proteins and the rabbit anti-6×His IgG antibody and rabbit anti-S.iniae serum,respectively,which indicated that these 5 recombinant candidate proteins were expressed successfully and with good immunogenicity,which providing the basic materials for the S.iniae subunit vaccine research and development.5.The immunoprotection effect of 5 recombinant proteins on channel catfish against S.iniae DX09Use rSrr,rNeuA,rK710,rFbpA and rFe as antigens to vaccinate the channel catfish,and detect serum antibodies titers by ELISA.Results suggested that the serum antibody levels of 5 groups were all elevated which indicated these 5 recombinant proteins could all stimulate channel catfish to produce corresponding antibodies,and the antibody levels of different groups reached the peak at different time after vaccination.The protective efficacy trials were conducted 28 days post vaccination with 14 days continuous observation.Results showed that the cumulative mortality rate of groups rSrr,rNeuA,rK710,rFbpA and rFe were 30%,45%,50%,30%and 45%respectively,and the relative percent survival?RPS?were 70%,55%,50%,30%and 50%,respectively.Above showed that rSrr has the highest immune protective effect and could be applied for subsequent vaccine development against S.iniae infection in channel catfish. |
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