Studies On Selection Of LOS 2C7 Epitopes And Immune Effect Of The Fusion Proteins With HBc

Gonorrhea pathogen is Neisseria gonorrhoeae. There are more than 60 million new cases every year. N.gonorrhoeae infection may lead to a series of diseases, such as mucosal inflammation (urethritis and cervicitis), and the invasion to the organs. Gonorrhea and HIV infection are also closely related. Gonorrhea can be treated with antibiotics, but with the emergence of resistant strains, controlling gonorrhea with drugs faces serious challenges. To control gonorrhea by vaccine maybe the most economical and effective method. But little is known about the immune response during gonococcal infection and the mechanism of repeated infections, there is still no available effective vaccine for gonorrhea.Therefore, the effective target selection is the main research direction for gonococcal vaccine. N. gonorrhoeae LOS 2C7 epitopes were as major vaccine targets to study in this study, including the following aspects:(1) Screening N. gonorrhoeae LOS 2C7 epitopes. First, the LOS of N. gonorrhoeae antigen was extracted by hot phenol method, as the ELISA coating antigen. Second, 7 2C7 epitopes of LOS was synthesized and coupled with KLH, and used to immune Balb / c female mice. Sera were separated in first three-week and every week post boosting immunization for ELISA testing and evaluation of antibody level. The higher antibody levels of epitopes were screened. Third, the different protective levels of epitopes were evaluated by human serum complement-mediated killing assay and the higher protective levels of epitopes were screened. The antibody levels were evaluted by ELISA, and the immunogenicity of epitopes was determined by bactericidal tests.(2) Construction of recombinant gene and prokaryotic expression vector. The three selected epitope genes were synthesized, and the epitopes were connected with the linker GSGGSG, repeated three times. The 79,80,81 amino acids genes of HBcAg were knockout by overlapping PCR method. The epitopes were inserted into the HBcAg gene MIR area. The constructed recombinant HBcAg epitope genes were expressed by prokaryotic expression system. The pET22b expression vector was chosen and BamHⅠand HindⅢrestriction sites were selected. The construction expression vector was identified by restriction enzyme digestion and PCR and sequencing. (3) Expression and purification of epitope and HBcAg fusion protein. The expression condition was optimized in order to facilitate purification and promote the expression of soluble fusion protein. Soluble expression could be well expressed at 18℃. Fusion protein was purified by Ni column chromatography, and quantitied by BCA method, then used to immunize animals.(4) The evaluation of the immune effect of fusion proteins. Female Balb/c mice were immunized with purified fusion protein and bleed in first two weeks and each week post boosting immunization. Sera were separated for ELISA testing and evaluation of antibody levels of fusion protein. Bactericidal test and challenge test were used to evaluate the protective effect of fusion protein in Balb/c mice. Because the normal Balb/c mice are not susceptible to gonorrhoeae, the Balb/c mice were subcutaneously treated with 0.5mg17-βestradiol two days before and after challenge, respectively. The protective effect of fusion protein was further evaluated.In summary, ELISA, bactericidal test, molecular biology techniques, challenge test were carried out in this study for screening N.gonorrhoeae LOS epitopes with HBcAg fusion protein. The results showed that the screened epitopes with HBcAg fusion protein produced a higher antibody levels and better protective effect, and the screened LOS epitopes maybe N. gonorrhoeae vaccine targets.