Eukaryotic Expression And Identification Of Porcine Circovirus Type 2 Cap Protein In CHO-K1 Cell Line

Porcine circovirus type 2(PCV2)is prevalent in pigs from all over the world,ang has close relationship with PMWS,PRDC,PDNS,CT and porcine reproductive system disorders.These diseases are collectively referred to as porcine circovirus-associated diseases(PCVD),which seriously affects the healthy development of the world pig industry.Currently,the known porcine circovirus includes three types,porcine circovirus type 1(PCV1),type 2(PCV2),and type 3(PCV3).Among them,PCV2 nucleocapsid protein Cap contains antigen-neutralizing epitopes,can produce specific antibodies against PCV2,is the main target protein of PCV2 subunit vaccine and serum diagnosis,and is widely used in practical production.The antibody detection methods of PCV2 mainly include ELISA,IPMA and IFA,among which the ELISA antibody detection method has the advantages of simple operation,simultaneous detection of a large number of samples,and is widely used by scientific research institutions and farms.Based on the CHO-K1 eukaryotic expression system,the recombinant plasmid pEE12.4-PCV2-Cap was constructed and transfected into CHO-K1 cells.By pressured screening and limited dilution,a monoclonal cell line capable of efficiently expressing Cap protein was obtained.The cell strain was suspended,acclimated,fermented and purified to obtain the target protein,and the immunogenicity was verified by Western blot and animal immunoassay.The results showed that PCV2-Cap protein could be stably expressed in CHO-K1 cells;the viable cell density during fermentation was as high as 6.0×10 ~6 cells/mL,the cell viability was above80%,and the PCV2-Cap protein production can get to 370 mg/L.The establishment of Western blot and indirect ELISA assay proved that the protein has good immunogenicity.In this study,a CHO-K1 suspension-stable cell line expressing PCV2-Cap protein was successfully constructed,and an indirect ELISA assay was established,which laid a foundation for the establishment of a porcine circovirus type2 serological assay.