Generation Of Recombinant Virus-Like Particles Of Porcine Circovirus Type 2 Capsid Protein In E.Coli And Immunogenicity Of The VLPs

Porcine circovirus type 2(PCV2)is closely associated with postweaning multisystemic wasting syndrome,reproductive failure,porcine respiratory disease complex,porcine dermatitis and nephrophthy syndrome,pneumonia,enteritisc and other PCV2 associated disease(PCVAD),which cause economic loss to the globle swine industry.Control measure for PCVAD largely depends on the use of PCV2 vaccines.Genetic differences exist among PCV2 isolates and PCV2 has been divided into different subtypes,PCV2 a to PCV2 e.The available commercial PCV2 vaccines contain either inactivated PCV2a/b whole virus particles or recombinant PCV2 a capsid protein.These preparations most likely contain varying amounts of immuneirrelebant proteins that can cause adverse injection site reactons,with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process.Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins.Hence,development of new PCV2 vaccines is necessary.Recombinant virus-like particles(VLPs)due to their repetitive surface epitopes,the virus-like structure and the capability to induce humoral and/or CTL responses are more suitable for the development of immunogenic vaccines.There were multiple attempts to produce recombinant PCV2 Cap protein in different expression systems.In most cases the cap protein was expressed as truncated or full length monomeric Cap protein.Expression of PCV2 Cap in insect cells resulted in recombinant protein selfassembled into VLPs that were structurally and antigenically indistinguishable from regular PCV2 capsids.However,due to the high cost of antigen preparation,the market price of vaccines prepared from PCV2 Cap VLPs is very high.Employing cheaper ways of preparation of PCV2 Cap VLPs as antigen for vaccines should be considered.In the current study,we demonstrate that PCV2 b derived Cap VLPs can be efficiently produced in E.coli.Moreover,we demonstrate that E.coli-derived recombinant PCV2 Cap VLPs represent a promising antigen for a PCV2 vaccine.The contents of isthis study are composed of the following four parts:1.Soluble expression,purification and identification of PCV2 Cap protein VLPs in Escherichia coliAccording to the codon preference of E.coli,the full-length optimized ORF2 gene of PCV2 was synthesized and cloned into pET-32a(+)expression vector.The resulted recombinant plasmid pET-32a-cap was transformed into E.coli BL21 host bacteria.Expression of Cap protein was verified by SDS-PAGE and Western-blotting.The results showed that the Cap protein was expressed in E.coli by a soluble form.Western-blot showed that an expected 27 k Da protein band was recognized by the anti-PCV2 serum antibody.Electron microscopy showed that expressed Cap protein was able to self-assemble into VLPs with a diameter of 17-20 nm,and the morphological structure was similar to that of PCV2 virus particles.The Cap protein VLPs was purified by using ammonium sulfate precipitation.2.Immunogenicity of PCV2 Cap VLPs in miceTo evaluate the immunogenicity of the expressed PCV2 Cap VLPs,different amout of purified Cap VLPs were emulsify with prepared Cap VLPs immunogen and commercial Cap VLPs subunit vaccine,whole virus inactivated vaccine of LG strain and adjucvant only,respectively.Blood samples were collected every week post immunization.All serum samples were tested by a Cap-based anti-PCV2 Ig G ELISA.T cellular immune responses were also detected by T lymphocyte proliferation assay.Results showed that anti-Cap antibody titers in mice immunized with Cap VLPs immunogen prepared in this study were higher than those of mice immunized with the commercial subunit vaccine and whole virus inactivated vaccine of LG strain.The proliferation activities of T lymphocytes from mice immunized with Cap VLPs immunogen prepared in this study were higher than those of mice immunized with the commercial subunit vaccine and whole virus inactivated vaccine of LG strain.3.Establishment and application of indirect ELISA antibody detection method based on PCV2 Cap VLPsBy using purified Cap VLPs as coating antigen,an indirect ELISA was established for detecting anti-PCV2 antibody.The optimum assay conditions were as follow: antigen coating amount of 1ug/well,the coated plates were placed at 37 for ℃1 h and then at 4 overnight;serum working dilution at 1:400,incubation time was ℃1.5 h at 37℃;dilution of secondary antibody at 1: 8000,TMB reaction time was 15 min.Samples were considered positive if the sample-to-positive(S/P)ratio was greater than or equal 0.2235,and were considered as negative if the S/P value was less than 0.1905 as negative.The developed ELISA showed good specificity and reproducibility and accuracy when compared with commerical anti-PCV2 Ig G detection kit.4.Immunogenicity of PCV2 Cap VLPs in pigsTo evaluate the immunogenicity of the expressed PCV2 Cap VLPs,different amount of purified Cap VLPs were emulsified with carbomer adjuvant to make immunogen.PCV2-negative 20-day-old piglets were immunized with prepared Cap VLPs immunogen and commercial Cap VLPs subunit vaccine,whole virus inactivated vaccine of LG strains and adjuvant only,respectively.Blood samples were collected every week post immunization.All serum samples were tested by a Capbased anti-PCV2 Ig G ELISA.The results showed that the antibody levels of pigs immunized with Cap VLPs immunogen prepared in this study were higher than those of pigs immunized with the commercial subunit vaccine and whole virus inactivated vaccine of LG strain at the 14 d and 28 d after immunization.In summary,PCV2 Cap VLPs were expressed in soluable form in E.coli.The purified Cap VLPs showed good immunogenicity in mouse and pig.The results of this study will lay a firm foundation for subsequent development and application of cheaper PCV2 Cap protein VLPs subunit vaccine.