Transcriptomic Analysis,dominant Antigen Screening And I-ELISA Development Based On Serogroup A And B Bovine Pasteurella Multocida

Hemorrhagic septicemia(HS),caused by Pasteurella multocida(Pasteurella multocida,Pm)is an acute,fatal and septicemic disease of cattle and buffaloes.At the end of 2016,two bacterial strains,named Pm1 and Pm2 were isolated from tissues of Haemorrhagic septicaemia suspicious dead cow collected from Beijing and Shandong via mice injection.They were identified to be capsular serogroup A and LPS type 3 Pm by colonial morphology,culture characteristics,biochemical reaction,16 S rRNA gene sequencing,Pm species-specific PCR,capsular serogroup A and B multiplex PCR,lipopolysaccharide(LPS)genotyping multiplex PCR(LPS-mPCR)and capsule A depolymerisation test.In order to investigate the differences between the two serotypes,in this study,two single nucleotide resolution transcriptom maps of the strain Pm1(serotype A)and strain CVCC448(serotype A)were analyzed by strand-specific RNA-seq.The results showed that 810 genes were significantly different among the homologous genes of the two strains,506 genes were down-regulated in group B strain compared with group A strain.The enrichment analysis of Gene Ontology(GO)showed that the main difference between group B and A strains was on the surface of cell membrane.Since many components on the surface of cell membrane play an important role in the invasion of bacteria into host,itimplied that the pathogenicity of the two subtypes of Pm might be variant.In addition,the different genes between the two serotypes were also widely existed in ABC transporters,indicating that the energy metabolism of the two serotypes wasalso different,and metabolic activities may affect the virulence of the bacteria.Based on the results of transcriptome sequencing,the dominant antigen of bovine Pasteurella multocida was screened by immunoprecipitation(pull-down).With the mass spectrometry and bioinformatics analysis,the outer membrane lipoprotein carrier protein Lolb was selected as a candidate antigen.According to the nucleotide sequence of the outer membrane lipoprotein carrier protein Lolb gene in GenBank(Gene ID: CP 003022 Range: 1141355 to 1141951),a pair of specific primers without signal peptide sequence was designed and synthetised.To clone and express the gene,pET28a-Lolb and pMALl-Lolb vectors were successfully constructed and expressed in E.coli BL21(DE3)strain.The His-LolB protein was presented as inclusion body while the MBP-LolB protein was detected in the supernatant by SDS-PAGE analysis.MBP-LolB recombinant protein was obtained from bacteria lysis by affinity chromatography.Western blot analysis showed that the recombinant protein could react with the bovine antisera against Pm,suggest that this recombinant protein could be dominant antigen of Pm.Furthermore,MBP-LolB protein wasemployed as coated antigen to establish indirect ELISA.The results showed that the optimal concentration of antigen was 0.125ug/ml,the optimal dilution of serum was 1:100 and the optimal dilution of HRP labeled rabbit anti-bovine IgG was 1: 10000.In conclusion,two strains of bovine Pasteurella multocida serotype A and LPS type 3 were isolated and identified as well as investigated by transcriptomics sequencing.Then dominant antigenic protein Lolb was obtained by differentiating capsular serotypes A and B An indirect ELISA method was established,which laid a foundation for the detction of Pasteurella multocida antibody of cattle.