Preparation And Identification Of Antibody Against Cap Protein Of Porcine Circovirus Type 2,3 And 4

Porcine circovirus type(PCV)has no envelope structure and is a very small ss DNA virus.Up to now,four types of porcine circovirus,namely PCV1-4,have been found.It is generally believed that PCV1 is not pathogenic,while PCV2 is pathogenic;PCV3 and PCV4 have certain pathogenicity,which need a lot of data or research to confirm.The structures of PCV1-PCV4 are similar.The genome contains two major open reading frames(ORF).ORF1 encodes replication related Rep protein and ORF2 encodes Cap protein.Cap protein is the main antigenic protein of the virus.It has the primary antigenic determinants of the virus.Compared with Rep protein,Cap protein has stronger immunogenicity and plays an important role in the process of virus transmission and infection.According to research reports,PCV1-PCV3 exists widely in the world,and PCV4 has also been found in China and South Korea,etc.,but PCV4 doesn't have sufficient research data limited to a short discovery time.As an immunosuppressive virus,PCV2/PCV3/PCV4(PCVs)can co-infect with other viruses and bacteria,resulting in huge losses of global pigs.Although several vaccines based on VLPs have been available for PCV2,which can significantly reduce virus infection and virus induced lesions.What is more serious is that the vaccines of PCV3 and PCV4 have not been commercially available.Nevertheless,PCV3 has been widely spread all over the world and can only be controlled by drugs at present.The global pig industry has always been facing great challenges.Thus,vaccine research and antibody development for PCVs are very important to control the transmission and infection of PCVs.The purpose of this study is to prepare anti PCV2/PCV3/PCV4 Cap protein(Caps)antibody and construct the prokaryotic expression plasmid of PCVs ORF2 to remove part of nuclear localization signal(NLS).Besides,the study also aims to optimize the IPTG induced expression conditions which realize the high-efficiency expression of Caps,and purify and obtain the recombinant caps by affinity chromatography.In order to obtain universal antibodies,rabbits with more extensive antibody lineage and better recognition of small molecular antigens than mice were selected as immune animals.Rabbits were immunized with linear Caps not assembled into virus like particles(VLPs),avoiding the problem that epitopes may exist in VLPs to avoid immunity.High immune serum was obtained after immunization,and cross protective reaction was found between the three antisera.By changing the IPTG induction conditions,the efficient soluble expression of Caps was realized,and Caps self-assembled to form VLPs;after the rabbits were immunized with Caps and VLPs in the same amount,the trivalent antibody against Caps was obtained;the egg yolk antibody(Ig Y)was extracted from the egg yolk of immunized hens,and the obtained Ig Y had high titer by agar diffusion evaluation.There are differences in structure and antigen between Ig Y and Ig G.Due to the advantages of high yield,easy access and high affinity,Ig Y can become a powerful tool for the study and diagnosis of PCVs.In conclusion,recombinant Caps were obtained by prokaryotic expression,and the recombinant protein was purified by affinity chromatography.High immune antiserum was obtained from rabbits immunized with linear Caps,and there was cross protective reaction between the three sera,which verified the existence of universal antibody.Rabbits and Hailan brown layers were immunized with VLPs to prepare trivalent antiserum and Ig Y and the use of i ELISA,two-way agar diffusion,WB,IFA and other experiments verified that the antibody has high titer,high specificity and high affinity.It also confirmed that the VLPs obtained in the study have the potential to be used as vaccines.