Molecular Investigation On Epidemiology Of Porcine Circovirus Type Ⅱ In Guangdong Province And Establishment Of ELISA Detection Apporaches

Porcine circovirus virus type2was a kind of infectious diseases whichseriously damaged the pig industry. It inhibited the body’s immune system, madeother pathogens infect host, and caused airframe secondary infection which made thecondition more complex and serious. It was popular in world and made hugeeconomic loss.There were different complete genome of PCV2strains which presentdifferent leading in different times and different regions. PCV2was awalysvaried.There were many methods to detect PCV2antibody especially ELISA. TheELISA method based on ORF2encoding protein was unable to distinguish betweenvaccine antibody and virus antibody.The study amplificated PCV2whole genome to find out the variation of PCV2.Meanwhile the study constructed prokaryotic expression plasmid pET32a-ORF3andsynthesised pET32a-ORF2, and established ELISA method based on purifiedrecombinant proteins to detect PCV2antibody which provided new detection methodsto diagnosis PCV2.In order to know the epidemic, variation and evolution of PCV2, serum samplesfrom suspected pigs infected PCV2in many farms of Guangdong province were usedto isolate PCV2. According to the sequence of PCV2published in GenBank,3pairsof primers was designed and amplified by PCR. The results showed that each of9genomes showed1767bp in length. The homology of9isolates were93.6%-99.4%,while the homology with isolates in2012were94.5%-99.7%, the homology withisolates in2011were94.2%-99.7%, and the homology with other isolates in GenBankwere92.5%-99.7%. The ORF1of the9isolates had94.9%-99.9%nucleotidesequence homology and95.3%-100%amino acid homology; The ORF2had89.0%-99.9%nucleotide sequence homology and86.4%-100%amino acid homology;The ORF3had92.4%-100%nucleotide sequence homology and86.8%-100%aminoacid homology. The amino acid of ORF1, ORF2, ORF3had27,35and24variationlocuses respectively. only ORF1and ORF3had more variation locuses than isolates in2011and2012.5isolates were belong to PCV2d genotype while3isolatesbelonging to PCV2b and1isolate belonging to PCV2a. The analysis of PCV2genomic characteristics isolated from2011to2013found that the nucleotidesequence of PCV2was stable, and the homology of these isolates were high withother isolates of the world. PCV2b and PCV2d were the influenza strains.According to the isolate YD01(YB2013) gene sequence, the study designed apair specific primers and amplificated ORF3gene by PCR, meanwhile constructed theprokaryotic expression plasmid pET32a-ORF3. the study optimized ORF2geneaccording to the codon Preference of E.coli and removed signal peptide, synthesisingthe prokaryotic expression plasmid pET32a-ORF2. With the purified recombinantprotein as envelope antigen, the study primarily established two indirect ELISAmethods to detect PCV2antibody. The ORF2recombinant protein best concentrationwas1.69ug/mL, and serum best diluted multiple was1:50while the best dilution ofEnzyme antibody was1:15000. The ORF3recombinant protein best concentrationwas0.51ug/mL, and serum best diluted multiple was1:200while the best dilution ofEnzyme antibody was1:10000. The variation coefficient of within-run assays andbetween-run assays were less than7%. The ORF2recombinant protein and ORF3recombinant protein didn’t react with other pig disease positive serum. Comparedwith commodity kits to test clinical samples, the coincidence rate of ELISA based onORF2protein was97.4%. ELISA based on ORF2protein could be used to detectvaccine antibody while ELISA based on ORF3protein could be used to detect wildvirus antibody in the vaccined serum.