| PCV2 is recognized as one of the most important viruses causing severe economic impact in the swine industry worldwide and it has been described to cause different conditions depending on the virus, host immunity, co-infections and other environment characteristics. These diseases are grouped under the name porcine circovirus diseases (PCVDs). In recent years, PCVDs occurred frequently and had caused severe economic harm to the swine industry in China. Detection of serum antibody was widely used to investigate the infection state of PCV2 and evaluate the immunity effects of the virus vaccine. Enzyme linked immunosorbent assay (ELISA) played an important role in the diagnosis and serological investigation of PCVD disease, because of its sensitivity, specificity, rapid detection, simplicity and large-scale application, etc. Research had showed that the domestic ELISA kits to detect the antibody against porcine circovirus type 2 were general in low sensitivity. The import kits were difficult to be widely used because of its high price. Therefore, to establish a rapid, simple, sensitive and specific diagnostic method is an important significance for the preventing and controlling against PCV2.PCV2 ORF2 gene amplified by PCR was cloned into pET-30a, and then transformed into E.coli BL21(DE3). By using the SDS-PAGE analysis, as well as ImageJ and Graphpad prism 5.0 software, the factors closely related to the expression of the recombinant Cap protein were optimized which were culture growth temperature, induction OD600, induction IPTG concentration and induction time. The optimized expression conditions of the recombinant Cap protein were that when the OD600 reached 1.0～1.5, adding IPTG to final concentration of 0.2 mM, and shaking 175 rpm for 5 h at 37℃. To obtain highly purified recombinant Cap protein, the purification conditions were also optimized to determine the concentration of imidazole. The research showed that the concentration of imidazole in the washing buffer was 100 mM and in the eluate was 300 mM. Western blot showed that the purified recombinant Cap protein could react with PCV2-positive sera but couldn’t with positive serum of E.coli BL21(DE3) and E.coli BL21(DE3) transformed with pET-30a(+) after induction. These results confirmed that the purified Cap protein had good immunogenicity and specificity.PCV2 Cap-ELISA method was established with the purified recombinant Cap protein as the coating antigen through optimizing the reaction conditions. The coating concentration protein was 3 μg/mL, incubated at 4℃ overnight. The optimal blocking buffer was consisted with 5% horse serum and 2.5% BD skim milk. The plates were blocked for 2 h at 37℃. Serum sample was diluted with 200× and incubated for 45 min at 37℃. Rabbit anti-pig IgG-HRP was diluted with 5000× and incubated for 45 min at 37℃. The color developing time was incubated for 15 min at 37℃. The ELISA cut-off value was 0.26 which was determined by using TG-ROC method. Thus, samples with S/P ratios≤ 0.26 were considered PCV2 antibody negative, and those with ratios> 0.26 were considered positive. PCV2 Cap-ELISA had no cross-reaction with the positive reference serum antibodies to PRVã€TGEVã€RV and PPV. The intra-assay variation coefficient ranged from 0.32% to 7.8%, whereas the inter-assay variation coefficient ranged between 1.09% and 7.71%, which showed a nice repeatability. In the comparative experiment with eight kind of commercial ELISA Kits, the coincidence rates of PCV2 Cap-ELISA were 89.22%,83.83% and 89.22% respectively which was consistent with three kind of imported kits. While the rates were uneven and they were 67.67%,80.23% ,54.49%, 65.87%，71.25% and 84.43% respectively, comparing with five kind of domestic kits. The research indicated that the detection results were reliable, and PCV2 Cap-ELISA could be used for the detection of clinical samples. |
