Study On Immunogenicity And Protective Efficacy Of Three Recombinant Proteins From Acinetobacter Baumannii

Objective:To explore a new strategy for recombinant subunit vaccine of Acinetobacter baumannii(AB),the recombinant OmpK,Omp22 protein and OmpK/Omp22 fusion protein of AB were obtained by recombinant expression.The immunogenicity and immunoprotection of three recombinant proteins were detected and compared after animal inoculation.Methods:1.PCR method was used to amplify OmpK and Omp22 gene by using AB ATCC19606 genomic DNA as templates,and OmpK/Omp22fusion gene was obtained by overlap extension PCR.OmpK、Omp22 gene and OmpK/Omp22 fusion gene were cloned into pColdI vector,and the obtained recombinant plasmids were identified by restriction enzyme digestion and DNA sequencing.The obtained recombinant plasmids were transformed into Escherichia coli BL21(DE3)respectively.After IPTG induction,the recombinant protein was purified on Ni~+-NTA column.2.Balb/c mice were randomly divided into four groups:Omp K,Omp22,OmpK/Omp22 immunized group and control group.Each mouse in the test group was immunized subcutaneously with 20μg corresponding recombinant protein in 50μL PBS formulated with an equal volume of Freund’s Complete Adjuvant,and boosted with the same dose of corresponding protein formulated with Freund’s Incomplete Adjuvant at day 14 and 21.Each mouse in the control group was injected subcutaneously with 50μL PBS plus 50μL Freund’s Complete Adjuvant for the first day and 50μL PBS plus 50μL Freund’s Incomplete Adjuvant at day 14 and 21.In the 7th and 21st days after the last immunization the blood of mice were collected through internal canthus vein,and antiserum titers were determined by ELISA.3.Spleen lymphocytes were isolated on the 21st day after the last immunization,and stimulated by the corresponding antigen in vitro,the proliferation of lymphocytes was detected by MTT assay and the ELISA method was used to determine the release level of IFN-γcytokines.4.Three weeks after of the last immunization,the mice were challenged with AB clinical isolates.The survival rate of the mice was recorded.The peripheral blood of the mice was collected to detect the bacterial load.The pathological changes of lung tissue of the mice were observed after HE staining.Results:ThreerecombinantplasmidspColdI-OmpK/Omp22,pColdI-Omp K and pColdI-Omp22 were successfully constructed and transformed into E.coli BL21.After induced with IPTG,recombinant plasmids can express three recombinant proteins OmpK/Omp22,OmpK and Omp22.The antibody titers of the OmpK/Omp22 immunized mice on the 7th and 21st day after the last immunization were significantly higher than those of the OmpK immunized mice or the Omp22 immunized mice(P<0.05).The rate of splenocyte proliferation of OmpK/Omp22 immunized mice was significantly higher than that of OmpK and Omp22 immunized mice(P<0.05).The production of splenocyte IFN-γin Omp K/Omp22,OmpK and Omp22 immunized mice were 0.521±0.069ng/m L and 0.48±0.042ng/mL,0.485±0.050ng/mL respectively,which were higher than those in PBS control group(0.423±0.1088 ng/mL).The mice in PBS control group all died with in 72 hours after infection with clinically isolated strains of Ab.The survival rates of mice immunized with OmpK/Omp22,OmpK and Omp22 until the8th day after infection were 66.7%and 25%,37.5%respectively.OmpK/Omp22,OmpK,Omp22 immunized mice peripheral blood bacterial load were lower than the PBS control group(P<0.05),while the mice peripheral blood bacterial load in OmpK/Omp22 fusion protein group was the lowest(P<0.01).The lung tissue of PBS control group showed severe interstitial pneumonia and a large number of inflammatory cells around the blood vessels.OmpK and Omp22 immunized mice showed moderate pneumonia and moderate inflammatory cells infiltration,while the lung tissue of the mice immunized with OmpK/Omp22 fusion protein showed only mild inflammatory reaction with very small number of inflammatory cells in the perivascular areas.Conclusion:Allthreerecombinantproteinsshowstrong immunogenicity and a certain degree of protective efficacy after immunization.The immunogenicity and protective efficacy of the fusion protein OmpK/Omp22 are significantly stronger than either of the single recombinant protein OmpK and Omp22.The results of this study can provide reference for further research on molecular design of subunit vaccine and construction of multi-subunit vaccine against AB.