Identification And Research Of Differential Genomic Genes Between Virulent And Avirulent Strains Of Bovine Pasteurella Multocida Capsular Serotype A

Pasteurella multocida is the causative agent of numerous diseases worldwide, diseases caused by P. multocida include haemorrhagic septicaemia in cattle and buffalo, enzootic bronchopneumonia in cattle, sheep and goats, fowl cholera in poultry, atrophic rhinitis in swine. Human infections with P.multocida predominantly occur following cat- and dog-inflicted injuries and even casual contact with pets. Five capsule serotypes are routinely identified in P. multocida（A, B, D, E, and F）. Based on the lipopolysaccharide for classification into serotypes 1¹6. Since 2008, it has been reported the first case that isolated P. multocida serotype A from cattle, which had caused secondary infection happened in several provinces and cities in our country, the substantial morbidity and mortality resulted in great economic losses to the development of live-stock breeding.P. multocida can be divided into virulent and avirulent strains depending on LD50,the key factors which influence strain pathogen are genes that encoding virulence factors or regulating expression of virulence genes. Comparison of the genome sequences can provide useful information of genes that are specific for highly virulent isolates. Genes in genome sequences of pathogenic strains but absent in non-pathogenic strains play important roles in pathogenesis. Studies on the antigens expressing by virulence genes showed good immune activity. Suppression Subtractive Hybridization is a mature technology applied on screening and identification different genes between virulent and avirulent strains, SSH make enrichment of different genes. We attempted to get different DNA fragments between high virulence strain Pm CQ2 and low virulence strain Pm CQ6. Combined the genome analysis results and selected the candidate genes, made the verification through PCR. Finally, we cloned different genes, and did deeply research on specific genes. Our research laid a solid foundation for study of P.multocida molecular pathogenic mechanisms.The main experimental contents and results are as follows: 1. Identification of differential genomic genes between virulent and avirulent strains of bovine Pasteurella multocida serotype A by Suppression Subtractive HybridizationTo study the differential genomic genes between virulent and avirulent strains of bovine Pasteurella multocida capsular serotype A, we used Suppression Subtractive Hybridization（SSH）to analyze the different genes between virulent strain Pm CQ2 and avirulent strain Pm CQ6. Following genomic subtraction and DNA sequencing, all of 28 different DNA fragments were obtained, the BLAST result showed that 27 DNA fragments coding proteins associated with virulence, metabolism, transcription and gene synthesis, 1 DNA fragments coding hypothetical protein. All these sequences showed high similarity with Pasteurella multocida. The novel differential genes provide a reference for follow-up studying the pathogenesis of bovine Pasteurella multocida capsular serotype A. 2. Bioinformatics and PCR analysis of differential genomic genes between virulent and avirulent strains of bovine Pasteurella multocida serotype A28 differential genomic gene fragments were screened through bioinformatics tools with the whole genome sequence of bovine Pasteurella multocida serotype A strain Pm CQ2 and Pm CQ6. We got Pm CQ2-6g0023, showed similarity to phage tail assembly protein I, had 30 bp sequence gene deletion compared with Pm CQ6-8g0001. We speculated phage genes were different between genomic genes based on the result of gene analysis. The distribution of 9 genes were further analyzed using PCR in 6 high virulence strains including Pm CQ2 、Pm CQ1、Pm CQ3、Pm CQ4、Pm CQ5 and Pm CQ7 of bovine Pasteurella multocida serotype A. The results revealed that these genes including Pm CQ2₂g0233、Pm CQ2₂g0235、Pm CQ2₅g0023、Pm CQ2₆g0001、Pm CQ2₆g0006、Pm CQ2₆g0018、Pm CQ2₆g0019、Pm CQ2₆g0021 were present only in 6 high virulence strains but absent in Pm CQ6. 7 genes were related with phage, this results proved the validity of the speculation. 3. Study of differential genomic gene Pm CQ2₆g0018 between virulent and avirulent strains of bovine Pasteurella multocida serotype AThe different genes were cloned, then ligated into the prokaryotic expression vector p ET-30a（+）, recombinant fusion proteins were induced by IPTG, the SDS-PAGE analysis showed four candidate molecular were successfully expressed in E.coli BL21. The recombinant fusion proteins r Pm CQ2-6g0018 and r Pm CQ2-6g0021 harboring histidine tag were abundantly purified by Ni-NTA Superflow Cartridges. The results of SDS-PAGE and western blot showed that r Pm CQ2-6g0018 was a molecular weight of about 19 KD, and with better immune activity.We studied gene Pm CQ2-6g0018 in lung of mice at transcription level, the result showed that at 16 h time point, compared with control the expression of Pm CQ2-6g0018 significantly increased.Purified protein r Pm CQ2-6g0018 was used to immune mice and antibody titer was determined by ELISA. The result demonstrated that the antibody levels in the immunized groups were significantly higher than which in the control group. Two weeks after the last time of immunization, 2LD50（4.4í105cfu） dose of bovine Pasteurella multocida serotype A strain Pm CQ2 was used to challenge mice through intraperitoneal injection. The results showed that mice in control group were all dead quicklly after challenged by bovine Pasteurella multocida serotype A strain Pm CQ2. The survival rate of mice in the immunized group of the recombinant proteins of r Pm CQ2-6g0018 was 25%. The protective rate of Pm CQ2 whole cell protein was 100%. The research on the phage tail gene of Pm CQ2 genome DNA will provide new direction for further study of the pathogenesis of bovine Pasteurella multocida serotype A...