| Pasteurella multocida(P.multocida)is a major bacterial disease in the Chinese pig industry,mainly causing swine pneumonia and atrophic rhinitis.Since there are many serotypes of P.multocida and the cross-protection between serotypes is still unclear,it is necessary to conduct more in-depth research on the antigen type and immune mechanism of P.multocida.In this study,four protective antigens,OmpA,OmpH,plpP and plpE,were selected as research objects,recombinantly expressed in vitro and formulated into subunit vaccines,and mice were used as experimental animals for immunological challenge protection experiments,in order to further explore and develop pig sources.The multi-killer Pasteurella subunit vaccine research provides data accumulation.1.In this experiment,four protective antigen genes of OmpA,OmpH,plpP and plpE were cloned using the PCR method of PmA8.1 strain DNA stored in our laboratory,and constructed into pET-28(a)vector.Four recombinant expression plasmids,pET28-OmpA,pET28-OmpH,pET28-plpE and pET28-plpP,were used to prepare recombinant proteins in vitro for subunit vaccination with aluminum gel adjuvant,Freund’s adjuvant and ISA201 adjuvant.The mice were challenged.The results showed that the four recombinant plasmids were transformed into E.coli BL21(DE3),and the expression of OmpA,OmpH and plpE were induced by inclusion bodies,and plpP was not expressed.In the OmpA vaccine group,the ISA201-OmpA attack protection rate reached 50%;the AL-OmpA and Freund-OmpA attack protection rate was 16.6%;in the OmpH vaccine group,the protection rate of AL-OmpH was 16.6.In addition,the protection ratios of the other two adjuvant groups were 0;in the plpE vaccine group,the ISA201-plpE challenge protection rate was 100%,and the protection rates of the other two adjuvant groups were 90%.2.Constructing four protective antigens of OmpA,OmpH,plpP and plpE and E.coli heat-labile enterotoxin type B subunit(LTB)fusion expression vector,and culturing the purified expression protein with adjuvant ISA201 to immunize mice.The results of challenge showed that LTB could enhance the immunoprotective effect of OmpH and plpE protein.The protection rate of LTB-OmpH fusion protein was 16.6%,and the protection ratio of OmpH constructed alone was 0.The LTB-OmpA fusion protein protection ratio was 16.6%,and the protection rate was lower than that of the antigen OmpA.The pplP protein protection ratio of the fusion expression was 33.3%.The protection ratio of the combined expression of OmpA,OmpH and plpP in fusion expression was 16.6%,which was equivalent to the protection rate of LTB-OmpA and LTB-OmpH alone.The protection ratio of the LTB-plpE fusion protein was 100%,and only 3 of the 6 mice developed symptoms.In summary,the best protective effect of this test is plpE.The protective effect of LTB-plpE and plpE subunit vaccines prepared by ISA201 adjuvant against PmA8.1 strain is 100%.The incidence of-plpE immunized mice was significantly lower than that of mice immunized with plpE.Followed by OmpA,the separately constructed OmpA and ISA201 adjuvants formulated with a vaccine have a 50% protective effect. |
