ABSTRACT
Objective:To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice.Methods: The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T.Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pOMPH was obtained.Then the recombinant plasmid was trans fected into SP2/O cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR,Westem blot analysis and indirect immunofluorescence.Three groups of BALB/c mice(n=16) named pOMPH,pCDNA3.1(+) and PBS were intramuscularly vaccinated with the recombinant plasmid,control vector and PBS respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation (SLP) and secreted IFN-γof spleen were tested by MTT.The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization,the protection rate were counted.Results: RT-PCR,Western blot analysis and indirect immunofluorescence showed that the omph gene could be,transfected into SP2/0 cells in vitro and expressed the target protein.Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups(P＜0.01).Spleen lymphocyte proliferation by MTT assay indicated that the SI value induced with avian Pasteurella multocida Omps in pOMPH group was higher than those in pCDNA3.1 (+) and PBS groups (P＜0.05).The IFN-γexperiments(Double-antibodies-sandwich-ELISA)showed that the levels of IFN-γ induced with Omps in the group of pOMPH was mostly higher than in the other control groups apperent(P＜0.01 ).The protection rate of pOMPH(70%) was better than in the other groups.Conclusion: The omph DNA vaccine against avian Pasteurella multocida had been constructed successfully.The DNA vaccine could enhance the immunity level and the protective effect of the vaccinated mice.Present study may be useful for the development of avian Pasteurella multocida vaccine.
ABSTRACT
Objective:To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice.Methods:The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T.Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pOMPH was obtained.Then the recombinant plasmid was transfected into SP2/0 cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR,Western blot analysis and indirect immunofluorescence.Three groups of BALB/c mice(n=16) named pOMPH,pCDNA3.1(+) and PBS were intramuscularly vaccinated with the recombinant plasmid,control vector and PBS respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation (SLP) and secreted IFN-? of spleen were tested by MTT.The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization,the protection rate were counted.Results:RT-PCR,Western blot analysis and indirect immunofluorescence showed that the omph gene could be transfected into SP2/0 cells in vitro and expressed the target protein.Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups(P