Preparation And Potential Application Of A High-titer Polyclonal Antibody Against Capsid Protein Of Porcine Circovirus Type ?

Porcine circovirus type 2(Porcine circovirus 2,PCV2)is one the key causative agents that leads to severe damage to pig industry worldwide.PCV2 infection can cause a variety of PCV2-associated diseases(PCVD),including post weaning multiple system failure syndrome,porcine dermatitis and nephrotic syndrome,porcine diarrhea syndrome,and reproductive disorders.PCV2 belongs to a single-stranded circular DNA virus that is one of the smallest known animal viruses.The virus preferentially targets the immune cells including macrophages,dendritic cells T cells and B cells,resulting in immunosuppression in pigs.In addition,PCV2 infection also facilitates the co-infection with various pathogens.Although commercial vaccines can effectively reduce occurrence of PCVD,the virus cannot be eventually eliminated in pigs.It was shown that infection rate of PCV2 is more than 60%in the pig herds in China.However,the pathogenesis of PCVD is not fully understood.Capsid protein(Cap),encoded by the ORF2 gene,is the major structure protein of PCV2,playing critical roles in viral infection and host-virus interaction.Hence,generation anti-Cap polyclonal antibodies is much convenient to detect PCV2 infection and to study the pathogenesis of PCVD.However,expression of full-length PCV2 ORF2 in E.coli is inefficient and most of the recombinant proteins are in form of inclusion bodies.In order to prepare an efficient polyclonal antibody against PCV2b Cap,truncated Cap protein without nuclear localization signal peptide containing a His-tag was expressed in E.coli expressing system.After optimization of the expression conditions,highly soluble recombinant Cap proteins were acquired and purified.New Zealand white rabbits were then immunized with the purified soluble recombinant Cap to generate antiserum.The anti-Cap IgG was further purified by caprylic acid-ammonium sulfate precipitation and the purity of IgG was examined by SDS-PAGE and coomassie brilliant blue staining.ELISA analysis showed that the titer of the purified anti-Cap antibody was 1:10~7.To examine the biological activity of the home-made antibody,a serial of diluted antibodies was applied to detect Cap proteins in PCV2b-infected PK-15 cells,mouse tissues and clinical tissue samples by immunoblotting,immunofluorescence(IF),or immunohistochemistry(IHC).The results showed that Cap antigen could be specifically detected in PK-15 cells and mouse tissues infected with PCV2b by IF and/or IHC.Additionally,viruses can also be specifically examined by the anti-Cap antibody in tissues infected with clinical PCV2b strains by immunoblotting.In summary,an efficient prokaryotic expression system expressing truncated PCV2b Cap protein lacking of NLS peptide was successfully generated in this study.The recombinant Cap proteins were soluble without affecting the its immunogenicity.Finally,high tier anti-cap polyclonal antibodies with highly specificity were prepared,which can be used for immunofluorescence,immunohistochemistry,immunoblotting,and detection of clinical PCV2b.Hence,our study provides a useful tool for diagnosis and research of PCV2b and PCVD.