Study On Brucella Double-antigen Polysaccharide Conjugate Vaccine

Background Brucellosis poses a greater threat to the health,economic development and biosecurity of the people in our country.Vaccine immunization is the main means of preventing and controlling Brucella infection.At present,the commonly used Brucella vaccine is live attenuated vaccine,but due to its safety problems,it is rarely used for human,and there is no internationally recognized Brucella vaccine for humans.The development of a safe and efficient new Brucella vaccine has become an urgent problem to be solved.Objective Preparation of Brucella O-antigen polysaccharide carrier protein conjugates in Yersinia enterocolitica using bacterial glycosylation system.Meanwhile,E.coli was utilized to express protein antigen omp19,and the two were further combined and physically mixed in vitro to prepare a novel subunit vaccine with both protein antigen and polysaccharide antigen.In addition,to improve the conservation and effectiveness of protein antigens,a screening identification of Brucella epitope peptides was performed to provide antigenic targets for subunit vaccine studies of Brucella.Methods 1.In the process of preparing Brucella polysaccharide conjugate vaccine using the binding of cholera toxin(CTA2 protein and CTB protein)in vitro,the Omp19 fusion CTA2 protein expression vector and CTB protein expression vector were constructed respectively.After purification,the proteins were obtained.The binding of CTA2 and CTB was performed using two methods: acid denaturation,alkali-urea renatural,acid denaturation-alkali renatural.2.In the process of preparing Brucella polysaccharide conjugate vaccine using the binding of Spy Catcher and Spy Tag in vitro,the Say Tag peptide tag fusion Omp19 plasmid was constructed and the protein was purified.At the same time,spycatcher fusion proteins already constructed in the laboratory were subjected to protein purification.Finally spycatcher and spytag proteins were mixed at different molar ratios in 4°C in 1 × PBS overnight to explore the optimal ratio of the two proteins.3.In the process of preparing Brucella polysaccharide conjugate vaccine by physical mixing,the Omp19 protein expression vector was successfully constructed.The Omp19 protein monomer and CTB-OPS glycoprotein were obtained by protein purification.Mice were injected subcutaneously after mixing the two proteins,and the first,second,and third immunizations were performed on days 0,14,and 28,respectively.The immune dose of Omp19 protein was 20 μg,and the immune dose of CTB-OPS was 2.5 μg.A rat tail blood collection was performed one week after each completion of immunization,and the serum was isolated the next day for Ig G antibody titer assay.Brucella infection is carried out on day 10 after the completion of the third immunization,and the dose of infection was the maximum amount of the pathogenic bacteria that is not fatal to mice.Spleen bacterial loading was performed on day 6 after infection,and point plate counting was performed.4.After Brucella abortus infection mice,the PMBC cells infected mice were isolated,the MHC-binding peptides were enriched by antibody immunoprecipitation,and the antigenic epitope peptides that could bind to mouse MHC in Brucella were identified by mass spectrometry.Results 1.In the process of preparing of Brucella double-antigen polysaccharide conjugate vaccine using the binding of cholera toxins in vitro,we successfully constructed the CTA2 fusion Omp19 protein expression plasmid,and purified to obtain a high purity protein.2.In the process of preparing of Brucella double-antigen polysaccharide conjugate vaccine using the binding of Spy Catcher-Spy Tag in vitro,we successfully constructed the Spy Tag tag fusion Omp19 protein expression plasmid,and was able to obtain the protein of interest after protein purification.Binding pre-experimental in vitro results showing that Spy Catcher-Spy Tag is feasible to build double-antigen polysaccharide conjugate vaccine.3.In the process of preparing of Brucella double-antigen polysaccharide conjugate vaccines by mixing double-antigen in vitro,we successfully constructed the Omp19 protein expression plasmid,and the corresponding protein of interest can be obtained after protein purification.When the immune antigen was glycoprotein,no other adjuvant,the survival rate in mice after lethal dose infection was the highest,at 80%.When the immune antigen is only protein antigen,the antibody titer produced after mixing with aluminum adjuvant was the highest,and the number of pathogens in the spleen of the mouse is the smallest.When glycoproteins were physically mixed with proteins,the survival rate of mice decreased by 60%.4.In the process of screening and identification of Brucella epitope peptides that can bind to mice MHC in Brucella were identified by mass spectrometry.Among them,19 peptides was related to MHC-Ⅰmolecules and 16 peptides was related to MHC-Ⅱmolecules.Conclusions In the research process of Brucella double-antigen polysaccharide conjugate vaccine,the method of in vitro binding using Spy Catcher-Spy Tag is feasible.Combined with mass spectrometry and bioinformatics analysis,35 epitopes of Brucella antigens were identified successfully.