| ObjectiveThe 30 proteins that may be present in the outer membrane selected from the whole genome of Neisseria gonorrhoeae FA1090 were screened by the research group in the early stage,and their T cell and B cell epitopes were analyzed,and the initial study of membrane localization and immunogenicity were carried out to further screen and evaluate its potential as candidate vaccines and molecular diagnostic targets.Methods1 Bioinformatics analysis: The physicochemical properties,hydrophobic properties and advanced structures of proteins of these proteins were analyzed using Prot Param tool,Prot Scale,SOPMA and SWISS-MODEL software,and homology analysis using BLASTp,B cell epitopes analysis using ABpred and Bepi Pred,CTL epitopes analysis using Net CTLpan and SYFPEITHI,Th epitopes analysis using SYFPEITHI and Net MHCIIpan.2 Cloning,expression and purification of candidate outer membrane proteins: By gene synthesis and recombination PCR method,recombinant plasmids containing the target gene were constructed,and transferred to the prokaryotic expression system and induced expression with IPTG,and purified the proteins by nickel column affinity chromatography.3 Preparation of candidate outer membrane proteins immune serums: the purified recombinant proteins were mixed with Freund's adjuvant and emulsified to immunize BALB / c mice,a total of two immunizations,and immune seria were obtained by eyeball extraction method.4 Preliminary study on immunogenicity analysis and membrane localization of candidate outer membrane proteins: Enzyme-labeled plates were coated with Neisseria gonorrhoeae sonicated whole bacterial protein,and then the Ig G antibody titer of each immune serum was detected by indirect ELISA to evaluate its immunogenicity;Enzyme-labeled plates were coated with Neisseria gonorrhoeae whole bacterial protein,and then the binding ability of each protein immune serum to whole cell antigen was detected by indirect ELISA to preliminarily evaluate whether the protein was expressed on the membrane surface.Results1 Through the analysis of the physicochemical properties and hydrophilicity and hydrophobicity of 30 candidate outer membrane proteins and the prediction of advanced structures,the results show that there are 7 proteins with stable physicochemical properties,good heat resistance,and rich antigen epitope,and the protein accession numbers are:YP?209068.1,YP?207267.1,YP?208542.1,YP?207398.1,YP?208811.1,YP?207981.1and YP?208090.1.Through homologous analysis and epitope prediction,26 proteins with high homogeneity were found,9 of which had more T-cell epitopes and B-cell epitopes,and the protein accession numbers were YP?208090.1,YP?208296.1,YP?208618.1,YP?208811.1,YP?208748.1,YP?208979.1,YP?207439.1,YP?207701.1 and YP?209122.1.2 15 recombinant plasmids were constructed,7 proteins were expressed in supernatant through different induction conditions,8 proteins were expressed in the bacterial pellet as inclusion bodies,and 15 purified proteins were successfully obtained by nickel column affinity chromatography.3 All candidate outer membrane proteins can induce specific Ig G antibodies against Neisseria gonorrhoeae after immunizing mice.The top one proteins with the highest titer were YP?208618.1,the lower efficacy is protein YP?208542.1,YP?209122.1 and YP?209068.1.4 The immune serum of each candidate outer membrane protein obtained after immunity mice was combined with the whole cell antigen of Neisseria gonorrhoeae,of which the difference between the antiserotonin of 13 candidate outer membrane proteins and the negative control group was statistically significant.ConclusionsThrough the Wayne analysis to integrate the bioinformatics analysis,it is found that YP?208090.1 and YP?208811.1 have the most abundant advantages of B-cell epitopes and advantageous T-cell epitopes,and have great potential as a target for molecular diagnosis and candidate vaccine molecular research.Experimental studies on the 15 candidate outermembrane proteins showed that YP?208618.1 had strong immunogenicity of induced Ig G antibodies.Preliminary experimental results of membrane localization show that 13 of the 15 predicted candidate outer membrane proteins are expressed on the outer membrane surface.Among them,the protein YP?208618.1 has relatively high immunogenicity,strong binding ability to whole cell antigens,and may have a high expression in the outer membrane of bacteria,so it has the greatest potential for becoming a vaccine candidate for Neisseria gonorrhoeae. |
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