Construction And Selection Of Antigen Epitopes Of Acinetobacter Baumannii Fribronectin-binding Protein Omp33-36 And TonB

Objective:The aim of this study was to screen B cell and T cell antigen epitopes of Acinetobacter baumannii Fribronectin-binding proteins Omp33-36 and TonB.Recombinant proteins were expressed and used to immunize mice for detecting their immune effects in vivo.A series of immunological methods were used to determine both B cell and T cell epitopes of Omp33-36 and TonB,which are preparations for developing monoclonal antibodies and epitope-based vaccine.Methods:B cell epitopes and T cell epitopes were predicted by bioinformatics methods and synthesized.Recombinant plasmid pET30a-omp33-36 and pET28a-tonB were cloned and converted into E.coli BL21 for induced expression.Then fusion protein was purified by affinity column chromatography,and analyzed by SDS-PAGE,identified by Western Blot.Following that protein were injected into Balb/c mice for three times at two week interval,besides the freund's adjuant was added for immune enhancement.Serum was collected from each of immunized mice and negative control mice then used to perform indirect ELISA with synthesized peptides to identify B cell epitopes.Cells of spleen were separated from every mouse and cultured with each of synthesized peptides respectively,then cell supernate was collected to detect IFN-?secretion by double sandwich ELISA for T cell epitopes selection.Results:1.Epitopes of Omp33-36 and TonB were predicted by bioinformatics methods.According to several bioinformatics methods,3 candidate B cell epitope peptides(named Omp_33-36B1-Omp_33-36B3),3 candidate T cell epitope peptides(named Omp_33-36T1-Omp_33-36T3)of Omp33-36 and 4 candidate B cell epitope peptides?named TonBB1-TonBB4?,4 candidate T cell epitope peptides?named TonBT1-TonBT3?of TonB were chosen and synthesized.2.Recombinant plasmid pET30a-omp33-36?pET28a-tonB was successfully constructed,Omp33-36?TonB was expressed and purified for immunized.Full-length of Omp33-36 gene and TonB gene was amplified from AB.genome and cloned into vector pET30a and pET28a respectively.PCR,double enzyme identification and sequencing results showed that construction was correct.Protein Omp33-36?TonB were expressed in E.coli upon induction by IPTG.SDS-PAGE result showed that Omp33-36 protein was about 34kDa and TonB protein was about34kDa.3.B cell epitopes of Omp33-36?TonB were identified by indirect ELISA.Indirect ELISA results showed that peptide Omp_33-36B1,Omp_33-36B2 reacted with Omp33-36 immunized mice's sera and peptide TonBB3,TonBB4 reacted with TonB immunized mice's sera.The OD₄₅₀nm values were significantly higher than those of PBS immunized mice?p<0.05?,which confirmed that those peptides were B cell epitopes.4.T cell epitopes of Omp33-36 and TonB were determined by double sandwich ELISA.Double sandwich ELISA results showed that Omp_33-36T3 stimulated splenocyte of Omp33-36 immunized mice produced significantly higher concentration of IFN-?than those of PBS immunized mice?p<0.05?.TonBT2,TonBT3 and TonBT4stimulated splenocyte of TonB immunized mice produced significantly higher concentration of IFN-?than those of PBS immunized mice?p<0.05?,which indicated that they were T cell epitopes.Conclusion:1.B cell epitopes and T cell epitopes of Omp33-36?TonB ptotein antigen were predicted by bioinformatics method.2.Prokaryotic expressing vectors pET30a-omp33-36 and pET28a-tonB were constructed successfully and Fribronectin-binding protein Omp33-36 and TonB were expressed and purified to immunize mice.3.Two B cell epitopes,one T cell epitopes of Omp33-36 were identified;Two B cell epitopes,three T cell epitopes of TonB were identified;This results lay a foundation for the development of Ab.Subunit vaccine and optimized recombinant DNA vaccine.