Prediction And Identification Of The B Cell Epitopes Of The PCV2 Cap Protein

Porcine circovirus is non-enveloped viruses,with a circular,single-stranded DNA genome.It is one of the smallest animal viruses.According to the pathogenicity,antigenicity and gene sequence of PCV,it can be divided into PCV1 and PCV2.PCV2 is considered to be the main pathogen of weaning multisystemic wasting syndrome.PCV2 also can cause mixed or secondary infection with a variety of other pathogenic,which increased the difficulty of disease precaution,and resulted in serious economical losses to the pig industy.Cap protein is the only structural protein of PCV2,which contains major neutralization epitopes and involved in the protective immune response,it is the preferred object for disease diagonsis and vaccine preparation.The aim of this study was to screen the B cell linear epitope of PCV2 Cap protein,which can provid theoretical guidance for the development of PCV2 detection regents and vaccines.In this study,the PCV2 KF gene sequence was obtained from NCBI,and the genotype of PCV2 KF strain was determined to be PCV2 b by homology-tree.The secondary structure,hydrophilicity,flexibility,accessibility and antigenicity of PCV2 Cap were predicted by DNAStar.B cell epitopes of Cap protein were prediceted by ABCpred and Bepipred1.0 web servers.6 potential B-cell linear epitopes in Cap were predicted to locate in the regions of 58-66,79-91,151-162,174-189,204-213 and 221-233 by multiple schemes.Cap protein gene was truncated according to the prediected results.The gene were amplified by PCR,and then subcloned into the expression vector pET32a(+)via BamH?and Hind ?.Ecoli BL21(DE3)plysS cells were transformed with the recombinant plasmid,and induced with IPTG.SDS-PAGE and Western-blot analysis showed that the truncated proteins were successfully expressed.B cell linear epitopes of Cap protein were screened with PCV2 positive serum,3 reactogenicity protein peptides(Cap77-95,Cap174-189,Cap221-233)were selected.Analysis of homology indicated that 3 segments were unique to Cap,which may be the specific epitopes of PCV2.Three-dimensional structure of Cap protein was obtained by homology modeling,and the spatial distribution of epitopes were predicted.The results showed that 3 epitope regions were located on the surface of protein,which was beneficial to the recognition of antibody.In this study,3 dominant linear epitopes are successfully screened by prediction and experiment,and they have well immune response,which pave the way for the development of PCV2 detection reagent and epitope vaccine.