| Porcine circovirus(PCV) is the smallest virus as known so far.It can be divided into two types:PCV-1 and PCV-2 according to the pathogenicity,antigenicity and nucleotide sequence. PCV-1 widely exists in pigs and pig-derived continuous cell lines.Animal infected experiments prove that PCV-1 has no pathogenicity,but it closely correlates with the health of pigs.It is necessary to study on this pathogen.PCV-2 is assolated with many diseases in pig herds,for example,postweaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex(PRDC) and congenital tremor(CT).All these diseases have made immensely economic losses to the pig industry in the world.In this study,specific primers were designed to amplify ORF2 genes of PCV-1,and then expressed the ORF2 genes in prokaryotic system.We purified the recombinant Capsid proteins of PCV-1 that obtained,and used them as coating antigens.The indirect ELISA was developed after that,which had a good specificity,sensitivity and stability.Three pairs of specific primers were synthesized according to the ORF2 sequences of PCV-1 pubulished in the Genbank.Three gene fragments of ORF2 were amplified by PCR according to PCV-1 templates.Then the amplified fragments were cloned to pMD18-T vector.After sequence analysis,the three fragments were directionally subcloned into the expression vector Pgex-6p-1, and then transfected to the expression host bacteria BL21.The recombination proteins were obtained,reaching to 29%of total bacterial proteins and the molecular weight was about 41Ku, 40Ku and 38Ku,respectively.Western blot analysis showed that all these proteins had antigenicity.The recombinant Capsid protein with the best antigenicity was used as coating antigen,and it didn't have cross reaction with PCV-2 standard positive serum.After optimizing the reaction conditions,an indirect ELISA was developed for the detection of PCV-1 antibodies in sera.In addition,we used this Capsid protein and the prepared Capsid protein of PCV-2 in our laboratory together,and developed another indirect ELISA that could simultaneously detect PCV-1 and PCV-2.The standard positive sera of PRRSV,CSFV and PRV were respectivly detected by the two ELISAs,the results showed that both of the two ELISAs had good specificity.92 serum samples were detected by the two ELISAs and the presence of antibodies were also detected by IPMA,as a result,the agreement rate was 93.5%and 96.7%,respectivly.The results of duplicate test showed that the coefficient of variation was less than 10%.227 serum samples that collected from partial regions in our country were detected by these two methods.75 serum samples were PCV-1 positive and the positive rate was 33.04%;69 serum samples were PCV-2 positive and the positive rate was 30.4%;7 serum samples were mixed infection... |
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