| Objective: To construct Chlamydi psittaci MOMP and MIP DNA vaccine-loaded BG,and analyze its immune activities and the ability of protection against C.psittaci infections,providing experimental basis for further development of vaccines against C.psittaci.Methods: A prokaryotic expression vector p ET 30a-MOMP was constructed,and the recombinant MOMP protein was induced and purified.The eukaryotic expression vectors pc DNA3.1(+)-MOMP and pc DNA3.1(+)-MIP were constructed and loaded into empty BG by calcium chloride method.The expression of MOMP and MIP in RAW 264.7 cells were tested by Western Blot.BALB/c mice were divided into eight groups,including PBS,empty BG,pc DNA3.1(+)-MOMP,pc DNA3.1(+)-MIP,pc DNA3.1(+)-MOMP BG,pc DNA3.1(+)-MIP BG,pc DNA3.1(+)-MOMP/MIP BG co-immunization and pc DNA3.1(+)-MOMP/MIP BG(Heterologous boost,He)and all mice were immunized by intramuscular injection for 4 times.ELISA were performed to test the level of specific Ig G from sera at one week after the last immunization.CCK-8 Cell Counting Kit was used to test the levels of splenocytes proliferation,and IFN-? and IL-17 secretion levels of splenocytes were tested with ELISA after stimulated with relevant antigens in vitro.One week after the last immunization,1×105 IFU of C.psittaci was intranasally infected mice.The changes in hair status,activity level and body weight were observed daily.At 4 and 10 days after infection,mice were sacrificed by cervical dislocation.The lungs of half mice were used for the evaluation of inflammatory pathology after H&E staining.The other half mice lung tissues were collected and homogenized,and used for C.psittaci IFU counting by Indirect immunofluorescence assay and IFN-? and IL-17 detection with ELISA.Results: The prokaryotic expression vector of p ET 30a-MOMP was successfully constructed and the the target protein approximately 48 KDa was analyzed by SDS-PAGE.The result of 1.5% agarose gel eletrophoresis showed almost no nucleic acid was present in E.coli JM109 cytoplasm after chemical osmotic pressure treatment.pc DNA3.1(+)-MOMP BG and pc DNA3.1(+)-MIP BG could expressed in RAW 264.7 cells which indicated that pc DNA3.1(+)-MOMP and pc DNA3.1(+)-MIP were successfully loaded into the BG.After immunization,the Ig G titer in mice sera,stimulation index(SI)of splenocytes and levels of IFN-? in pc DNA3.1(+)-MOMP BG,pc DNA3.1(+)-MIP BG,pc DNA3.1(+)-MOMP/MIP BG co-immunization and pc DNA3.1(+)-MOMP/MIP BG heterologous boost mice groups were much higher than these in PBS,empty BG,pc DNA3.1(+)-MOMP and pc DNA3.1(+)-MIP mice groups(P<0.05).And there were significant difference between co-immunization and single immunization groups,also between co-immunization group and heterologous boost group(P<0.05).While there was no obvious difference in levels of IL-17 A between all groups were observed(P>0.05).On day 4 after C.psittaci infection,the degree of lung pathological changes or the IFU in the lung homogenate in pc DNA3.1(+)-MOMP BG,pc DNA3.1(+)-MIP BG,pc DNA3.1(+)-MOMP/MIP BG co-immunization and pc DNA3.1(+)-MOMP/MIP BG heterologous boost mice groups was significantly lower than that in PBS,empty BG,pc DNA3.1(+)-MOMP or pc DNA3.1(+)-MIP mice groups,co-immunized mice were lower than the single immunized ones,and heterologous boost group were also lower than the co-immunized group(P<0.05).On day10 post-infection,almost no Chlamidia were detected in all mice lung tissues,and the lung pathology showed chronic inflammation.Conclusions: The pc DNA3.1(+)-MOMP BG and pc DNA3.1(+)-MIP BG were able to induce a strong immunity response to produce a protective effect against C.psittaci infection.And the combination of MOMP and MIP could enhance its protective effects than single immunization.Heterologous boost could enhance the immune response of DNA vaccine loaded BG. |
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