Establishment And Application Of Indirect ELISA For The Detection Of Porcine Circoviurs Type 3 Antibodies

Porcine circovirus 3(PCV3)can cause multiple organ inflammation in pigs,porcine eermatitis nephropathy syndrome(PDNS),and reproductive disorders in sows.ELISA is an important tool for disease diagnosis,epidemiological investigation and immune effect evaluation.The capsid(Capsid,Cap)protein encoded by ORF2 has good immunogenicity and is an important target protein for vaccine and detection technology research.In this study,an indirect ELISA for PCV3 antibody detection based on Cap protein was established,which was used to detect serum samples collected from 2013 to 2020,and then the study analyzed the prevalence of PCV3 through the detection results by ELISA.This study aims to provide technical support for PCV3 detection,epidemiological investigation and prevention and control.Firstly,recombinant expression plasmids of PCV3-Cap protein were constructed through connecting PCV3-Cap protein gene sequence(without nuclear localization sequence)with prokaryotic expression vector pET-28a(+).And then the recombinant plasmids were transformed into Escherichia coli,and the recombinant protein with molecular weight of 25.4 kDa was expressed under IPTG induction.The expressed protein was identified via SDS-PAGE,Western bloting and Liquid chromatography-tandem mass spectrometry(LC-MS/MS).The results showed that recombinant PCV3-Cap protein was successfully expressed,the amino acid sequence of the protein was 88.4%consistent with the reference sequence,and the predicted epitopes were completely consistent with those of the reference strain.The recombinant protein could stimulate mice to produce specific antibodies,the average antibody level≥1:12800,indicating that recombinant Cap protein had good immunogenicity.Furthermore,a PCV3 indirect ELISA assay was established for detection of specific antibody by using recombinant Cap protein as antigen.When the OD450nm value of the serum to be tested was greater than 0.38,it was judged as positive for PCV3 antibody,while the OD450nm value was less than 0.38,it was negative for the specific antibody.The ELISA method established had no cross-reaction with porcine antibody positive serums such as PCV2,CSFV,PRV(gI and gB)and PRRSV.The coefficient of variation of in-batch and inter-batch repeatability test ranged from 1.30%to 7.14%.Finally,1562 serum samples from pig farms in different areas from 2013 to 2020 were detected by indirect ELISA.The results showed that in 2013,28%of sera were positive for PCV3 antibodies,and 26%of samples were double positive for PCV2 and PCV3 antibodiws.After that,the positive rate of serum PCV3 antibodies gradually increased,with the highest detection rate(94%)in 2017.At the same time,the detection rate of double-positive serum for PCV2 and PCV3 antibody double positive sera was also the highest.Subsequently,the detection rate of PCV3 antibody positive serum and the double positive rate of PCV2/PCV3 antibodies decreased,but still more than 50%.The PCV3 antibody detection rate of boars,through production and sows was as high as 90%～100%,the positive rate of PCV3 antibody of sick suckling and nursing pigs were higher than that of pigs without disease.