Prediction And Experimental Screening Of The Epitope Of PorB And NspA Of Neisseria Gonorrhoeae

ObjectivesTo analyze the secondary structure and dominant B cell and T cell epitopes of the outer membrane proteins PⅠand Nsp A of Neisseria gonorrhoeae by using Bioinformatics software, and The polypeptide were synthesized.Then the synthetic peptides were filtrated through the methods of Lymphocyte proliferation assay and ELISA test, To be specific, polypeptide reacted to lymphocytes and serum from the mice who was immuned with Neisseria gonorrhoeae antigen, polypeptide can stimulate lymphocyte proliferation well or the antibody titer high as candidate epitopes;Train of thought and a theoretical basis was provided for Neisseria gonorrhoeae epitope vaccine. Methods1 Advantages of B cells、T cell epitope peptide of neisseria gonorrhoeae outer membrane protein prediction: Por B protein choose the Neisseria gonorrhoeae standard strain CMCC 29403 nucleotide sequence of our own lab, conversed it into amino acid sequence by DNAStar software, a total of 348 amino acid residues; Nsp A protein is reported in Gene Bank WHO-A plant, A total of 174 amino acid sequence. Application online tools TMHMM to analyze the full-length amino acid sequences transmembrane region of Por B and Nsp A, then used tools GOR4 and SOPMA on EXPASY server to predict their secondary structure. Moreover, the flexibility, hydrophilicity, surface accessibility and antigenicity of Por B and Nsp A protein were analyzed via the Karplus-Schulz method, Kyte-Doolittle method, Emini method and Jameson-Wolf method of the Protean module of DNAStar software. According to the above results, B cell epitopes of Por B and Nsp A protein were predicted comprehensively. When T cell epitopes of Por B and Nsp A were predicted, the most common HLA genotype were selected, and the restriction of H2-Ed, Ek in mouse were considered too. The polypeptide scores were calculated by its amino acid on whether the anchor residues, auxiliary residues or advantage residues, and high scores peptides were chosen as the candidate T cell epitopes, and antigenicity of peptides were analyzed by the affinity between peptides and MHC molecules as the basis of judgment for Net MHC-II software, and higher affinity is strong antigenicity peptides. Comprehensive results of SYFPEITHI and Net MHC-II software were integrated to determine the possible regions of T cell antigen epitope.Predicted epitope peptides were synthetised by the company.2 Screening advantage of epitope peptides by experiment: Used formaldehyde treatment method、ethanol method and heating method to prepare whole-cell antigen of neisseria gonorrhoeae, to determine the best whole-cell antigen preparation methods under microscope after gram staining. Chose the optimal method to prepare inactivated whole cell antigen, immuned BALB/c mice after sterility testing, lymphocytes in spleen and lymph nodes and immune serum have been collected from mice, the proliferation degree of peptides on lymphocyte was measured by lymphocyte proliferation test, preliminary test was done to detect the antigenicity of polypeptides; Antibody titer of polypeptide on mice immune sera was measyred by ELISA to detet the ability of affinity between the polypeptide and the specific antibodies; Based on the above,screened the advantage of epitope peptide with good antigenicity. Results1 The transmembrane regions of Por B protein were located on its N-terminal of the 1-20 amino acids, the transmembrane regions of Nsp A protein were located on its N-terminal of the 1-25 amino acids, the rest amino acids located on inner membrane;The secondary structure of Por B and Nsp A protein was based on random coil,there is also the alpha helix and beta sheet. Synthesized its flexibility, hydrophilicity, surface accessibility and antigenicity, the advantage B cell epitopes of Por B protein may exist in the N-terminal amino acid sequence of the168~178, 127~133; the advantage B cell epitopes of Nsp A protein may exist in the N-terminal amino acid sequence of the 90~99,70~76.The advantage of T cell epitopes of Por B were possibly present in that of 212~226, 229~243, and the advantage of T cell epitopes were possibly present in 129~137, 2~16 of Nsp A by prediction of SYFPEITHI and Net MHC-II software.2 Microscopy results sμggest: Ethanol method preparde the best quality of the whole cell antigen, followed by formaldehyde, the antigen quantity by heat treatment was few, cell integrity has been destroyed, with the heating temperature increasing, the bacterium Neisseria gonorrhoeae antigen quality worse. Lymphocyte proliferation experiment measured that Nsp A protein B cell epitope 91~99, Por B protein B cell epitope 168~178 could effectively stimulated spleen lymphocytescells from mice immuned by whole-cell proliferation, Nsp A protein T cell epitope 137 ~ 130、2 ~16, Por B protein T cell polypeptide 212~226、243 ~ 229 amino acid sequence could effectively stimulated lymph node cells from mice immuned by whole-cell proliferation, ELISA tests showed that Por B protein B cells of 168~178, Nsp A protein B cells of 91~99 amino acid sequence,especially Por B protein B cells of 168~178 has stong ability of affinity to specific serum immuned by whole-cell antigen neisseria gonorrhoeae. ConclusionSuccessfully predicted the outer membrane protein por B and Nsp A T cell and B cell epitope of Neisseria gonorrhoeae.Lymphocyte proliferation experiment measured that the stimulation index of Nsp A protein B cell epitope 91-99, Por B protein B cell epitope 168~178 could effectively stimulated the spleen specific lymphocyte proliferation; Nsp A protein T cell epitope 137 ~ 130、2 ~16, Por B protein T cell polypeptide 212~226、243 ~ 229 amino acid sequence could effectively stimulated lymph node specific proliferation;ELISA tests showed that Por B protein B cells of 168~178, Nsp A protein B cells of 91~99 amino acid sequence had stong ability of affinity to specific serum immuned by whole-cell antigen neisseria gonorrhoeae. Therefore, 130~137 and 2~16 amino acid sequence of Nsp A protein, 229~243 and 212~226 amino acid sequences of Por B protein can be used as indominant T cell epitopes of Neisseria gonorrhoeae vaccine, 168~178 amino acid sequence of protein Nsp A, 91~99 amino acid sequence of protein Por B, In particular 91~99 amino acid sequence of protein Por B can be used as advantage of B cell epitopes of gonococcal vaccine.The results of this study provide new idea and theoretical basis for the subsequent development of Neisseria gonorrhoeae epitope vaccine.