Expression And Purification Of Porcine Circovirus Type 3 Cap Protein And Preparation Of Polyclonal Antibody

Porcine circovirus 3(PCV-3)is a new type of porcine circovirus that has been discovered in recent years.It can cause a variety of diseases in pigs in the clinic,causing serious harm and huge loss to the economy of the global pig industry.Nowadays,the research on PCV-3 in the world is still in its infancy,and the detection method is limited to the level of molecular biology.It is incapable of large-scale diagnosis and prevention of PCV-3,resulting in a gap in commercial ELISA test kits and vaccines.Researchers from all over the world are stepping up their research on PCV-3.At present,it is reported that the important structural protein Cap encoded by the PCV-3 ORF2 sequence has good immunogenicity and can induce an immune reaction.It can be used as an antigen to develop ELISA test kits,which are useful for distinguishing PCV-3 that is similar with PCV-2,PNDS and other diseases in clinical symptoms.At the same time,this method will facilitate mass detection and epidemiological investigation of PCV-3 on large-scale pig farms.In order to efficiently express PCV-3 Cap protein,the PCV-3 ORF2 sequence was used as a template to optimize the rare codons in the sequence(Shanghai Biotech)at the first time,and primers were designed for the optimized sequence to amplify 625 bp of target fragment.The target fragment was digested with the His-tagged p ET-28 a vector plasmid,and then ligated into a recombinant plasmid,transformed into an E.coli expression strain,and the positive colonies were picked and induced to express by enzyme digestion and sequencing.SDS-Page and Western Blot showed that the ORF2 gene was expressed in E.coli and the target protein size was about 27 k D.However,due to its low expression level,by site-directed mutation of the rare codon on the sequence,the best codon was obtained and the front-end repetitive amino acid sequence was cut off,reinducing expression,the amount of the target protein was greatly increased.Then the protein was treated by inclusion body,chromatographic purification and gelatinization purification steps to obtain a high-purity,high-concentration protein of interest.The purified protein was renatured and verified by colloidal gold immunoblotting.After negative staining and transmission electron microscopy,a "transparent" high-brightness circular substance was observed,ranging in size from 17 nm to 20 nm,which were consisitent with the size and morphology of the PCV-3 virions,it was judged to be PCV-3 virus-like particles,indicating that the protein was successfully renatured by dialysis.The renatured protein was used as the immunogen to prepare the antigen.After three immunization experiments,the serum containing the polyclonal antibody was obtained.The serum was used as the primary antibody and identified by Western Blot and ELISA.The polyclonal antibody in the serum was confirmed to have a good specificity.The molecular biologic,morphological and serological studies confirmed that PCV-3 Cap protein has good reactogenicity and immunogenicity,and the prepared polyclonal antibody has good specificity.This experiment laid the foundation for the preparation of PCV-3 ELISA kits.