RESUMO
Avian Pasteurella multocida is the causative agent of fowl cholera, a disease much affecting the poultry industry. In order to study the efficacy of the recombinant subunit vaccine constructed with ptfA gene of avian P. multocida, the ptfA gene fragment amplified by PCR from avian P. multocida was cloned into the prokaryotic expression vector pET32a and the recombinant plasmid pET32a-ptfA was obtained. The pET32a-ptfA was expressed in Escherichia coli BL21 [DE3] and the target protein rPtfA was purified. The purified protein was then mixed with Freund's adjuvant and the recombinant subunit vaccine was obtained. Three groups of chickens labeled as rPtfA, attenuated live vaccine and PBS were vaccinated with the recombinant subunit vaccine, attenuated live vaccine and PBS, respectively. Serum antibodies, peripheral blood lymphocyte proliferation [PBLP] and interferon-gamma [IFN-gamma] level secreted by peripheral blood lymphocyte were tested. The immunized chickens were finally challenged with virulent avian P. multocida and the protection rate was counted. Indirect ELISA showed the levels of antibodies in rPtfA and attenuated vaccine groups were most significantly higher than the other groups [P<0.01], and the former was slightly lower than the latter. Peripheral blood lymphocyte proliferation experiments and IFN-gamma experiments indicated that SI value and the levels of IFN-gamma induced by ConA in the two vaccine groups were significantly higher than those of the PBS groups [P<0.01], and that the attenuated vaccine group was higher than the rPtfA group. The protection rates of rPtfA and attenuated live vaccines were 45% and 75%, respectively. The results indicated that the PtfA recombinant subunit vaccine was capable of improving the immunity level and inducing a protective effect for the vaccinated chickens, but it was barely satisfactory
RESUMO
Avian Pasteurella multocida is an agent of fowl cholera. The protective effect achieved through orthodox vaccines is not ideal. The research on novel vaccines against avian Pasteurella multocida is imperative. In this study, the genes encoding outer membrane protein H and A [OmpH and OmpA] were cloned into the eukaryotic expression vector pcDNA3.1[+] and the recombinant plasmids, namely DNA vaccines [pOMPH and pOMPA] were obtained. Five groups of chickens [n=20 per group] were intramuscularly injected with the two recombinant plasmids, attenuated live vaccine, control vector pcDNA[3.1[+]] and PBS, respectively. The immune responses and protective efficacy were evaluated after immunization by serological and challenging. A significant increase in serum antibody levels was observed in chickens vaccinated with the attenuated live vaccine and the two DNA vaccines. Additionally, the lymphocyte proliferation [SI values] were higher in chickens immunized with the attenuated live vaccine and the two DNA vaccines than in those vaccinated with pcDNA[3.1[+]] and PBS [P<0.05]. Furthermore, the two DNA vaccines provided partial protection to the vaccinated chickens; however, the protective efficacy was inferior to that provided by the attenuated live vaccine