| Porcine circovirus type 2(PCV2)virus mainly infects weaned piglets at 6-18 weeks,resulting in suppression of the body's immune system and a mixed infection of multiple pathogens,Causing the occurrence of porcine circovirus association disease(PCVAD),it has caused huge losses to the pig industry.Vaccines are an effective means of preventing and controlling PCV2 infection.PCV2 has 11 potential open reading frames(ORFs),among them,ORF2 encodes the only structural protein(Capsid,Cap)of PCV2,with 233 or 234 amino acid residues and a molecular mass of about 28 k Da,which is an ideal target antigen for vaccine development.Now,domestic and foreign scholars have done a lot of work on PCV2 vaccine research,and have developed a variety of commercial vaccines based on PCV2a/b subtypes,mainly inactivated vaccines,subunit vaccines,chimeric vaccines,but the prices are high,and the transformation of the PCV2 epidemic subtype is currently taking place,so it is necessary to develop a inexpensive and effective PCV2 subunit vaccine.Characteristics of using small molecule ubiquitin-related modification tags to promote soluble expression and increase expression level of proteins,according to the codons usage of E.coli,the optimized cap gene of PCV2b(Gen Bank: AY969004.1)was synthesized,Two B cell epitopes of PCV2(195HVGLGTAF202?226LKDPPLNP233)were individually and/or tandemly conjugated at the C-terminal of Cap by PCR technology.The cap gene conjugated with PCV2 B cell epitopes were constructed into the p E-SUMO expression vector and transformed into the expression strain BL21(DE3)for expression.After verification by SDS-PAGE and Western blotting,the three chimeric proteins were correctly expressed.after optimization of protein expression conditions,at 26 ?,the final concentration of the inducer was 0.5 mmol/L,and the culture was induced for 8 h,most of the target protein was expressed in soluble form.The three chimeric proteins were purified by affinity chromatography and tags were removed by SUMO protease.The three unlabeled chimeric proteins(Cap-e1,Cap-e2,Cap-e12)could self-assemble into virus-like particle in the assemble solution,the diameter is around 23 nm and lightly larger than virus-like particles formed by ?NLS-Cap.Afterwards,the three chimeric proteins were emulsified in equal volume with the corresponding Freund's adjuvant,Twenty 6-week-old female Balb/c mice were randomly divided into 5 groups,each mouse is immunized with 30?g/100?l,at the same time,setting up PBS group and vaccine group as negative control and positive control,Immunize once every 2 weeks for 3 times,blood was collected at the 2nd,4th and 6th week,PCV2 antibody detection kit was used to detect the change trend of antibody,comparing the impact of different B-cell epitopes linked to the C-terminal on the immunogenicity of Cap,antibody test results show that,except for the second week,the antibody level of each test group was significantly higher than PBS group,and the antibody level produced by the Cap-e12 protein conjugated with 2 epitopes was higher than that of the protein linked only 1 epitope(Cap-e1,Cap-e2),in the sixth week,two weeks after the third immunization,antibody level of Cap-e12 group was continued to rise and had no significant difference with the vaccine group.The neutralization results measured by IPMA showed that the neutralization ability of Cap-e12 was significantly higher than that of Cap-e1 and Cap-e2.In summary,the conjugation of two epitopes can significantly improve the immunogenicity and neutralizing activity of Cap.This provides a certain reference for the development of a new PCV2 subunit vaccine. |
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