Development And Preliminary Application Of An Indirect Elisa For Detection Of Porcine Circovirus Type2Antibody

Porcine circovirus type2(PCV2) is the primary causative agent of post weaning-pig multisystemic wasting syndrome (PMWS) and other associated transmissible diseases. It can cause serious inhibition to porcine immune system and can easily lead to the infection of other diseases in pigs. At present, PCV2has become one of the most important infectious agents in pig industry, and has resulted in huge economic loss in the pig industry. However, there is no effective drug to prevent and control PCV2-related diseases. Therefore, to control the incidence and prevalence of PCV2in China is especially important. Because of the higher cost of antigen preparation of PCV2, the commercial PCV2ELISA kit price is also high. So a convenient, sensitive, specific and cheap serological method for large-scale detection of PCV2antibody should be established.The ORF2gene of PCV2is the main region encoding a viral capsid protein (Cap) that is the unique structural protein of the virus particle and also the major immunogenic protein.The Cap protein was considered to be type-specific antigen and it can be used to differentiate PCV1and PCV2. Therefore, it is especially meaningful for diease diagnosis and vaccine development. In this study, the Cap protein without PCV2nuclear localization signal (dCap), which was experessed by the small ubiquitin-like modifier (SUMO) fusion and the Escherichia coli expression system, was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of PCV2serum antibody.1. Cloning and expressing of PCV2dCap gene in Escherichia coli The coding region of PCV2dCap gene was amplified by PCR from virus genome. PCR product of dCap gene was cloned into prokaryotic expression vector pCold-SUMO which contained a SUMO tag and6×His tag to construct recombinant expression plasmid pCold-SUMO-dCap. The recombinant plasmid was then transformed into Escherichia coli Artic expression cells. It was induced by IPTG at15℃and active fusion protein of SUMO-dCap was efficiently produced. SDS-PAGE analysis of the recombinant protein demonstrated that soluble SUMO-dCap in the supernatant was expressed at approximately50%of total recombinant fusion proteins. Then, the soluble SUMO-dCap was purified by Ni-NTA Resin Purification Kits, and the SUMO tag was removed by the SUMO protease. In addition, the purification effect and specificity of the recombinant fusion protein and dCap protein were detected by Western blotting assay.2. Development and application of an ELISA assay for detecting PCV2serum antibodiesThis study used purified SUMO-dCap fusion protein as the coating antigen to establish an indirect ELISA for detecting PCV2serum antibodies. Simultaneously, the SUMO protein was employed as the control. The reaction conditions of ELISA were optimized. It was shown that the optimal protein concentration for coading96-microwell was0.938μg/mL, and incubated overnight at4℃. The plates were subsequently blocked with1%BSA-PBST for2h at37℃, and then incubated with diluted serum sample (1:160) for30min at37℃. The plates were washed three times and the diluted HRP-labeled goat anti-swine IgG (1:4000) was added and incubated for30min at37℃. Then the plates were washed three times again. Finally, substrate of TMB solution was added and incubated at room temperature for10min away from light to develop color. The threshold for ELISA was OD450nm≥0.272for positive, OD450nm≤0.233for negative, and the other was suspectives. The OD450nm value of SUMO control experiment were all less than0.233.267serum samples originated from pigs in different farms were assayed with this established ELISA method for validation and commercial ELISA kit was employed as control for comparison. The specificity, sensitivity and coincidence rate of the established ELISA method were93.33%(70/75),96.50%(171/176) and96.02%, respectively. A cross-reactivity assay demonstrated this ELISA method was PCV2-specific. Repeatability tests showed that the coefficient of variation of serum samples within and among runs were1.25%to6.37%and6.68%to13.58%, respectively. The results suggested that this ELISA was simpler to produce and perform. In addition, it was a specific, sensitive and convenient method for routine serodiagnosis and epidemiological survey of PCV2-associated diseases and evaluation of serum neutralizing antibodies against PCV2at low cost. It served as base for developing a PCV2ELISA diagnostic kit.