Comparison Of The Immunogenicity Of Recombinant Fragments Of Toxigenic Pasteurella Multocida Toxin

Toxigenic Pasteurella multocida(T+Pm) is a necessary causative pathogen of progressive atrophic rhinitis(PAR) in swine which is characterized by facial deformity, twisting of the snout and nasal hemorrhage. It is a widely prevalent, contagious swine respiratory disease that causes growth retardation and a reduction in the efficiency of feed utilization among grower-finisher pigs, which is also responsible for signif icant economic losses in the swine industry. Vaccination has been considered the most desirable and effective approach for controlling the disease. The Pasteurella multocida toxin(PMT) produced by P.multocida is the main virulence factor to induce PAR. It is a poor antigen and becomes more immunogenic after its native structure has been destroyed. Partially truncated proteins have been predicted to be good antigens.According to the gene sequences of Pasteurella multocida toxin in Gen Bank, PCR primers were designed. Then the gene of PMT was amplif ied from the genomic DNA of T+Pm HN-06 strains by PCR, and successfully cloned into the p MD18-T vector. After the prediction of the PMT secondary structure by DNAStar software, eight fragments were chosen to be the materials for the immunogenicity study of PMT. These fragments were amplified from p MD18-T-PMT by PCR, and then cloned into the high-expression vector p ET-32a(+), respectively. These recombinant expression vectors were successfully transformed into Escherichia coli BL21(DE3) being confirmed by PCR, enzyme digestion and sequence reaction. Using IPTG induction, all of them were expressed efficiently confirmed by SDS-PAGE. And their expressd products were mainly inclusion bodies. But only PMT1-506 aa, PMT1-391 aa, PMT505-1285 aa and PMT973-1285 aa were confirmed to react specifically evidently with the antiserum against native PMT by Western-blotting. The four fragments did not have lethal activity to mice proved in toxicity test. Then we chose three fragments which had the better reactogenicity with the antiserum against PMT to optimize the expressed conditions for higher productions.The PMT1-506 aa, PMT1-391 aa, PMT505-1285 aa and PMT973-1285 aa were processed as four vaccines and were used to immunize mice subcutaneously twice at 2-week intervals, respectively. The immunized mice were subcutaneously challenged with native PMT 2 weeks after the second immunization. The immunization and challenge study results showed that the survival rates of PMT1-506aa(10/10), PMT505-1285aa(10/10)and PMT973-1285aa(8/10)were significantly higher than that of PMT1-391aa(1/10)when challenged with 2 MLD of PMT, the survival rates of PMT1-506aa(10/10) and PMT505-1285aa(10/10)were significantly higher than that of PMT973-1285aa(3/10)and PMT1-391aa(0/10)when challenged with 5 MLD toxin, the survival rate of PMT505-1285aa(10/10)was significantly higher than that of PMT1-506aa(3/10)when challenged with 10 MLD toxin. Therefore, the PMT505-1285 aa group showed the highest survival rate of the four fragments.According to the result of simmunization and challenge study in mice, PMT505-1285 aa was processedas vaccine and was used to immunize weaned pigs intramuscularly twice at 2-week intervals. The immunized weaned pigs and control pigs were intranasally challenged with HN-06 strains of T+Pm(2×1010 CFU) two weeks after the second immunization. Thirty days after intranasally challenged, all of five weaned pigs(5/5) had the turbinate autopsy in the control group, while only one of five weaned pigs(1/5) did. Simultaneously,serum samples were collected from every weaned pig at before immunization and 2, 4 weeks post immunization. The antibody titers were measured using an indirect enzyme-linked immunosorbent assay(i ELIS A). The result showed that none of five weaned pigs(0/5) became positive in the control group, while three of five(3/5) became positive after first immunization and f ive of all(5/5) became positive after second immunization in the immunized group.Therefore, PMT505-1285 aa showed the best immune effect and could be a candidate for a vaccine against PAR.