| Pasteurella multocida is the causative agent of several diseases including haemorrhagic septicaemia in cattle and buffaloes, septicaemic/pneumonic pasteurellosis in sheep and goats, fowl cholera in birds, atrophic rhinitis in pigs and snuffles in rabbits, which ofen causes major economic losses to the live-stock industry around the world. Although there are many commercial inactivated vaccines and attenuated vaccines for preventing Pasteurella multocida at present, the disease has not been effectively prevented due to several reasons, such as many serotypes of Pasteurella multocida, the low immunogenicity between different serotype strains and short vaccine protection period. Bovine Pasteurella multocida serotype A, which causes bovine pneumonia, has been detected in some provinces of china since 2008. Because there is no available vaccine for serotype A inour country, it is very important for researchers to find newsecurity vaccineswhich can provide cross-protection against different types of Pasteurella multocida.With the development and applications of genomics and proteomics technologies, the research of vaccine science comes into a new era. Now, an increasing number of pathogenic microorganisms has been completed the sequencing of the whole genome, which provides data platform for the reverse vaccinolog to find new types of vaccines. Based on the genome sequence, reverse vaccinology opened up a new approach and has been increasingly applied to the development of new vaccines. By analysing pathogen genome sequence, reverse vaccinology is able to forecast the potential candidate immunogenic antigens. And there is no need to cultivate a large number of pathogenic microorganisms, just to high-throughput expression, then use Western blot or indirect ELISA and other immunological technology to analyse, screen and identifya specific antigen, and detecte the immune protection of candidate antigens in a mouse model, the screened protective antigen can provide a theoretical basis for the development of effective vaccines. This approach not only improves the success rate of screening candidate vaccines, but also helps to predict and discover unknown variety of immunogens, which has a very important and far-reaching significance.According to the idea of reverse vaccinology, the main experimental contents and results are as follows:1. Screening novel protective antigens of bovine Pasteurella multocida serotype A,and cloning, expression of themThe whole genome of bovine Pasteurella multocida serotype A strain PmCQ2 has been sequenced. We have been completed sequence analysis, gene annotation and the construction of PmCQ2 local strain genome sequence database. In this study we made use of the reverse vaccinology approach to predict and analyse proteins of PmCQ2, which contains Signal peptide and transmembrane domain using bioinformatics tools such as SignalP-3.0 server, SMART, and TMHMM Server v.2.0. And then fifty-six protective antigens candidates related to immunogenicity were screebed, the candidate genes were amplified by PCR after primers were designed, and the PCR products were digested with endonuclease, ligated in to pET32a(+) vector. E.coli DH5a and BL21(DE3) cells successfully transformed with recombinant pET32a(+) vector. Finally 56 candidates were successfully expressed.2. Screening antigen candidate moleculars of bovine Pasteurella multocida serotype A by indirect ELISAHarvested cell pellet which has been inducible expressioned and purified by Ni-NTA superflow cartridges. Our previous work has been successfully expressed 36 putative protein of bovine Pasteurella multocida serotype A strain PmCQ2and stored at-80℃ after purification. All these ninety-twoproteinswere dialyzed and coated onto the ELISA plate. Four kinds of bovine serum (including serotype A artificial infection bovine serum, serotype A inactivated vaccine serum, serotype B artificial infection bovine serum and serotype F artificial infection bovine serum) were used to screen proteins by indirect ELISA. The result of indirect ELISA showed that only 2 proteins interacted with artificial serotype A infected bovine serum but not with serotype B and F, 13 proteinsresponed toartificial serotype A infected bovine serum but not to serotype A inactivated vaccine serum, and the reaction of 20 proteins onartificialserotype A, B, F infected bovine serum.3. Immunogenicity of protective antigen candidate molecules in miceTo detecte the immune protection of the screened candidate antigens in a mouse model, the four recombinant proteins (1,11, L16, L17) were used for immunizing mice. The proteins were mixed with Freund’s complete adjuvant and immunized mice by subcutaneous route, respectively. There are 2 gradient groups (50μg and 75μg)for detecting the immune protection of the 4 proteins. At 10 days after the third time of immunization,3LD50 dose of bovine Pasteurella multocida serotype A strain PmCQ2 was used to challenge mice. The results showed that the control group were all dead quicklly after challenging, and the survival rates of the four proteins were more than 50%. To NO.11, the survival rates of 75μg(90%)is higher than 50μg(70%). After challenged by Pasteurella multocida serotype B and F strain. The results showed that the survival rates of NO.1, NO.11 and NO.L17 proteinsagainst serotype B were more than 60%, while the survival of them against serotype F is 0%, and the survival rates of NO.L16 is very low, which in keeping with the result of indirect ELISA.All the results indicated that 92 genes related to immunogenicity were selected by reverse vaccinology, and be detected the immunity protective in a mouse model. The results showed that NO.1, NO.11 and NO.L17 proteins can provide cross-protection against bovine Pasteurella multocida serotype A, B strain.These three proteins can be used as effective vaccines against bovine Pasteurella multocida, and provide a basis for screenning new targets for Pasteurella multocida. |
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