Preparation Of BN-Conjugated Porcine Circovirus Capsid Protein And Research On Its Bioactivity

Background:Porcine circovirus-associated disease(PCVAD)has brought serious economic losses to the world's pig industry and is one of the three infectious diseases in the pig industry.A capsid protein of porcine circovirus 2(PCV2)encoded by ORF2 gene serves as a diagnostic immunogenic antigen for the detection of PCV2-associated disease.Objectives:The main purpose of this study is to develop subunit vaccine based on capsid protein.The contents of experiment:We first couple h-BN with PCV2 capsid protein and study their biological activity.The main subjects of the experiment are as follows:(1)Construction of the expression system of capsid protein in Escherichia coli.(2)Purification of capsid protein.(3)Conjugation of h-BN and capsid protein,detecting the product by various methods.(4)Preparation of conjugated products and various adjuvant vaccines and immunizing mice,detecting of antibody titers and cytokine concentrations.Results:In this article,the recombinant plasmids(BL21(DE3)-pGEX-ORF2)containing capsid protein were transformed into E.coli.The capside protein cannot be detected by SDS-PAGE after induction.Since the N-terminal nuclear localization sequence(NLS)of ORF2 gene is rich in continuous rare codons,the expression of ORF2 gene is difficult in E.coli.Since the histidine tag is at the N-terminus and C-terminus of the recombinant protein,the target protein is isolated and purified by nickel ion affinity chromatography.Expand the culture of the expressed bacteria(BL21(DE3)-pET32a-ORF2),ultrasonically disrupted bacteria,and the supernatant was collected by centrifugation.The supernatant is purified by nickel ion affinity chromatography,and the target protein is purified successfully,detected by SDS-PAGE.The h-BN nanoparticles were characterized by XPS,SEM,TEM and FTIR.It was known that h-BN was disc-shaped particles with diameters of 30-60 nm.The zeta potential of h-BN dispersed in water was determined to be-30mV by laser nano-particle size analyzer,indicating that h-BN was very stable in water.The surface of h-BN nanoplates is first pre-treated with ammonium hydroxide and h-BN is coupled to BSA via glutaraldehyde successfully,detected by UV-Vis spectra and SEM.The coupling product BSA @ BN is successfully prepared.The BSA-loading capacity for h-BN nanoplates is 19%.In other words,the cross-linked BSA measured 0.19 mg per 1 mg h-BN.BSA @ BN was placed in different pH buffer,taken out at different time points,and the supernatant was collected by centrifugation to detect the protein release.BSA @ BN was found at acidic or neutral environment,less protein released into solution;in alkaline,more protein release.Aluminum hydroxide adjuvant vaccine,white oil adjuvant vaccine,gold nanoparticle vaccine and BN adjuvant vaccine(conjugated product of h-BN and capsid protein)were successfully prepared.ICR mice were immunized twice with these vaccines.Serum was collected.The concentration of IFN-y and antibody titer in serum was measured by ELISA.The results showed that BN adjuvant vaccine stimulated mice to produce a good immune effect.Conclusion:In this study,h-BN nanoparticles were first applied to the study of porcine circovirus subunit vaccine,and BN adjuvant vaccine was successfully prepared,which had significant immunological effect.