| Porcine circovirus(Porcine circovirus, PCV), a member of the Circovirus in Circoviridae, can be divided into two genotypes: PCV1 and PCV2. PCV1 has no pathogenicity; but PCV2 can cause the Postweaning multisystemic wasting syndrome(PMWS),which is an important immunosuppressive disease, and cause serious economic loss to the pig industry. There is no effective way to control and eliminate PCV2 so far, the vaccine on the market can not play a good effect. Therefore, it is very important to develop a serological detection methods to know prevalent condition of PCV2 and to take precautionary measures. The paper used the prokaryotic expression protein of ORF2 as antigen to establish the indirect ELISA assay and apply it to PCV2 serum antibody clinical detection.Firstly, a pair of special primers according to the complete genome of PCV2 HBEZ was designed to amplify truncated ORF2 gene, Sac I and Hind III were used in the primers. Truncated ORF2 gene fragment was recombined to vector p ET-28 a to construct recombinant plasmid p ET-ORF2. PCR and restrict enzyme identification confirmed that the recombinant plasmid were constructed successfully. Then the p ET-ORF2 was transformed into E.Coli Rosetta-gami p Lys S. After the adding of IPTG, the recombinant protine which was 34.5KDa was expressed, and was expressed in the form of inclusion bodies in the E.coli. The best induction conditions were: cultivate the bacteria to the OD value between 0.4 and 0.6 in 37℃, and then add IPTG to 1.0 mmol/L, the expression of protein was maximum after induced for 5h, could be 0.46mg/m L. The Cap protein was purified by the Ni-NTA Purification system and examined by Western-blot. The results showed that truncated recombinant protein could react with polyclonal antibody against PCV2, sharing a good reactionogencity.Secondly, using the purified proteins as coating antigen to develop an indirect enzyme-linked immunosorbent assay(ELISA). The optional conditions were determined.It was shown that the optimal concentration of the Cap protein was 4.6ug/m L, coating time was 37℃ for 2 hours and 4℃ for one night; 5% SMP/PBST was used as the best confining liquid and confined at 37℃ for 2 hours; 1:40 was the best dilution of serum sample and the reacting time was 1 hour in 37℃; The best concentration of HRP-labeled goat anti-swine protein Ig G was 1:3 000. The detection should be 37℃ for 1 hour and then chromogenic with TMB for 15 mins. Determined by statistical analysis of the critical value was 0.421. The specificity and repeatability of this method were evaluated. The result showed that the indirect ELISA established have no cross reaction with the positive serum of CSFV,PRRSV,FMDV,PRV,TGEV and PEDV. The repeated tests confirmed the variation of the test results were less than 8.71%. Prepared with other PCV2 antibody test kit, positive rate was 96.7%. Using this method to examine 223 serum samples got a result that positive rate was 61.9%(138/223); the positive of the piglets was 58.3%(63/108) and the growing pigs was 65.2(75/115). The result showed that the developed indirect ELISA could be used to detect the porcine serum antibodies to PCV2 and found the base of developing a ELISA diagnostic kit of PCV2. |
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