|
Abstract
Porcine circovirus 2 (PCV2) is a small, non-enveloped virus with a single-strand circular DNA genome that has had severe impact on the swine industry. Currently, two main genotypes of PCV2 have been recognized: PCV2a and PCV2b. PCV2b has been more frequently isolated from pigs with PCV-associated diseases (PCVAD) and may be a more virulent subtype than PCV2a.
The purpose of this study was to generate antibodies anti ORF2 protein (capsid protein) of PCV2b for developing diagnostic tools which can detect PCV2b antigens. BALB/c mice were immunized with capsid peptides of PCV2b. Mice were subsequently euthanized and spleen cells were fused with myeloma cells. Hybridoma supernatants were removed and screened for the presence of PCV2b-specific antibodies by an enzyme-linked immunosorbent assay (ELISA). After repeatedly cloning by limiting dilution and screening by ELISA, stable hybridomas secreting anti-PCV2b monoclonal antibodies (MAbs) were obtained and characterized. Eight clones secreted the IgM class of antibody, whereas six clones have the IgG1 isotype. Five MAbs produced positive signals with PCV-2 FA Substrate Slide (VMRD®).
|
|
Abstract
Swine vesicular disease (SVD) is a contagious disease of pigs, characterized by vesicles on the coronary bands, heels of the feet and occasionally on the lips, tongue, snout and teats. Swine vesicular disease virus (SVDV) is a member of the genus Enterovirus and family Picornaviridae. The virus is antigenically related to the human coxsackievirus B5. SVD is a notifiable disease and is strictly controlled in European countries by stamping out and restriction of pig movements. According to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, SVD antibody detection is a prescribed test for international trade. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to swine vesicular disease virus. Full length of VP1 and VP2 genes of SVDV were amplified by RT-PCR and the restriction enzyme cutting sites of BamHI and HindIII were added in the both ends of primer site. The RT-PCR amplicons were cloned into TOPO vector by a TA cloning kit and then the sequences were confirmed by DNA sequencing techniques. The target genes were subclone into the pET28, pET32, SUMO and pCold expression vector individually. The expression plasmids were transformed into the BL-21(DE3) host cell. IPTG is used to induce expression of cloned genes and the solubility of recombinant proteins was tested. Results showed that only pCold vector expressing products belonged to soluble protein. His taq protein purification method and gel extraction method were used to compare the purity of recombinant proteins. Results showed that gel extraction method could obtain recombinant protein in high purity. The purified recombinant protein of VP1 was coated in ELISA plates and indirect ELISA method were used to test a serial dilution of pig serum which obtained from the experimental SVDV infected pig. Results showed that linear curve could be observed in the indirect ELISA assay.
|