| Porcine circovirus type 2(PCV2) is a novel virus of the Circoviridae family was considered the cause of Postweaning piglets Multisystemic Wasting Syndrome (PMWS) which clinically characterized by wasting and death of postweaning pigs.PCV2 also been associated to a number of pathological conditions of pigs, including porcine dermatitis and nephropathy syndrome, reproductive failure, porcine respiratory disease complex, proliferative and necrotising pneumonia and congential tremor type A II. The wide spreading of the disease posed serious threat to pig industry and huge loss in many nations and regions in the world. The more serious thing was that the infection of PCV2 can depress the pigs immol/Lune system and lead to the second infection of other pathogens. Then, the more huge loss was made. Now the infection come from PCV2 has become one of the pathogens that can handicap progress of pig industry seriously. So far, there were not any good ways to control the infection of PCV2. The purpose of this experiment was to develop a good diagnostics way by molecular method.The paper was divided into 3 parts. Firstly, the expression of ORF2 gene of type 2 Porcine Circovirus in E.coli; Secondly, depurated and refolded ORF2 protein, and then use the refolded protein to establish the indirect ELISA method; Thirdly, We immune rabbits and Balb/c mice with the refolded ORF2 protein to obtain the polyclonal and monoclonal antibody. The result was described as follow:1. Primers were designed according to the sequence of GD strain. The ORF2 gene was amplified about 580bp use the primers, and then the gene was subcloned into prokaryotic expressing vector pET-32a. After that, the recombinant plasmids were transformed into Escherichia coli BL21-ΔE3 and induced by L-Arabinose. The results of SDS-PAGE indicated that the ORF2 gene was expressed and it is about 28ku. The quantity of the expression protein is about 30% of the E.coli protein.2. We purify the PCV2 ORF2 protein and we don't find any else protein cingulums detected by SDS-PAGE. Then the purification protein was refolded according to the Merck protein refolding kit. Western blot demonstrated that the refolded protein can be recognized by PCV2 polycolonal antbody.3. Establish the indirect ELISA method by using the refolded ORF2 purification protein. We optimize the reactivity condition through fumbling the concentration of antigen and the enzyme labeled antibody, the dilution of antibody and the reactivity time of every step. The results demonstrated that optimal concentration of recombinant ORF2 protein for coating was 0.5mg/L, the optimal coating condition of recombinant ORF2 protein for ELISA was incubated at 4℃overnight, the dilution of serum sample was 1:160, the work concentration of HRP-labeled rabbit anti-porcine IgG was 1:7500, the best incubation period and temperature for both the serum sample and the HRP-labeled rabbit anti-porcine IgG were 37℃for 45min, the substrate for ELISA was incubated at 37℃for 10min before terminated with the stopping solution. Meanwhile, interdiction test revealed that the indirect ELISA by the purified recombinant ORF2 protein has a good specificity for the detection of PCV2 antibody in sera. The coefficient of variation among wells in the same plate for ELISA was 2.78%, and there was no significant difference among plates, indicating that this ELISA assay established here had a good reiteration.4. We immune rabbits and mice by using the refolded ORF2 purification protein to product polyclonal and monoclonal antibody. The maximal dilution of the polyclonal of rabbits and mice against PCV2 ORF2 was 1:1.28×104 and 1: 107 mensurated by ELISA. We also gain positive hybridoma that product antibody against PCV2 ORF2. The two higher OD of the positive orifices were separately 0.734 and 0.781.
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