Evaluation Of The Immunogenicity And Protection Of Acinetobacter Baumannii Trivalent Fusion Protein Candidate Vaccine SmpA/Omp22/NlpA_1-128

Objective:To construct a trivalent fusion protein SmpA/Omp22/N1pA(1-128)of Acinetobacter baumannii and evaluate its immunogenicity and immunoprotective effect in Balb/c mice.Methods:1.The plasmid pcDNA3.1-SON which constructed by Sangon Sangon Bioengineering Company was used as template,primers were designed to amplify fusion gene fragment containing the complete coding sequence of the SmpA and Omp22 genesand the 384 base pair 5’end of the N1pA gene.2.The fusion gene was cloned into plasmid vector pCold I to construct recombinant plasmid pcold I-SmpA/Omp22/N1pA(1-128)。The recombinant plasmid was transformed into Escherichia coli BL21 for prokaryotic expression,and the recombinant protein was purified.3.36 female Balb/c mice were randomly divided into three groups:Recombinant protein immunized group,PBS treated group and adjuvant group.Each mouse in the recombinant protein immunized group was subcutaneously injected with 20μg recombinant fusion protein(in 100μL PBS)mixed with freund’s adjuvant in the same volume.Each mouse in the PBS treated group was subcutaneously injected with 100μL PBS buffer solution.And the mice in the adjuvant group were injected subcutaneously with 100μL Freund’s complete adjuvant.Each mouse in the three groups was boosted once with the same treatment of the first injection on 14th and 28th days after the first immunization.4.6 mice in each group were taken blood from the inner canthus.The serum was separated and ELISA kit was used to detect the antibody titer.Six mice in each group were sacrificed after the last blood collection.Then the spleen lymphocytes were isolated under aseptic conditions.After antigen stimulation,the spleen lymphocyte proliferation activity was measuredwith the CCK-8 kit,and the secretion of IFN-y and IL-4 were measured.5.The remaining 6 mice in each group were challenged with Acinetobacter baumannii.After 24 hours of bacterial challenge,blood sample was collected from the tail vein to detect the bacterial load,and the blood was collected from the endocanthus and the serum was separated to detect inflammatory factors;After 48 hours of challenge,the lung tissues of mice were isolated for pathological analysis.Results:The recombinant plasmid pCold I-SmpA/Omp22/N1pA(1-128)was successfully constructed.After transformation into E.coli BL21,a recombinant protein with molecular weight of 56kD was expressed and purified.The recombinant protein was identified by SDS-PAGE and Western-blot.Mice immunized with recombinant protein induced high levels of antigen-specific IgG antibodies.The proliferation activity of splenic lymphocytes and the level of IL-4 of mice in the recombinant protein immunization group were significantly higher than that of the PBS treated group and the adjuvant group(P<0.001),while the level of IFN-y in the three groups of mice had no significant difference;24h after the Acinetobacter baumannii challenge,the bacterial load of the mice in the recombinant protein immunizedgroup was significantly lower than that of the two control groups(P<0.001).The levels of IL-6 and IL-10 inthe serumof the recombinant proteinimmunizedmice were lower than those of the PBS and adjuvant mice after challenge(P<0.01).The lung tissue of the PBS treated group and the adjuvant group had obvious pathologicalchanges,while the lung tissue of the recombinant protein immunized mice showed very slight pathological changes.Conclusion:The trivalent recombinant fusion protein SmpA/Omp22/N1pA(1-128)induces a potent immune response and good protection after immunization in Balb/C mice.These results provide valuable data for further design and development of therapeutic and preventive vaccines against Acinetobacter baumannii.