| Porcine circovirus type2(PCV2) is the main and primary causative agent ofPostweaning Multisystemic Wasting Syndrome (PMWS), which has causedsignificant economic losses in the swine industry. The genome of PCV2is1767bp or1768bp in length and consists of11ORFs. Three major ORFs have beencharacterized for PCV2: ORF1, ORF2, and ORF3. ORF1encodes a replication-associated protein (Rep), while ORF2encodes a major structural protein (Cap), themain antigenic determinant of the virus, and ORF3is involved in PCV2pathogenesis.Cap protein contains several immune-dominant epitopes and is confirmed to be type-specific.To date, several commercial PCV2vaccines were under use in the market for theprevention and control of PCV2. Therefore, a method for specific serologic detectionin routine field practice to monitor PCV2antibody titers induced by vaccines isessential. Immunoperoxidase monolayer assay (IPMA), indirect immunofluorescentassay (IFA), and enzyme linked immunosorbent assay (ELISA) are the mostcommonly diagnostic methods for detecting PCV2antibodies. However, thesemethods require specialized equipment and technical expertise, and are suitable forlaboratory use only. Furthermore, IPMA and IFA require the cultivation of PCV2onPK-15cell, which is time-consuming and labor-intensive.The membrane-based immunochromatographic lateral flow strip test represents awell-established and appropriate technique for a variety of point-of-care andfield-use applications, which is used in the detection of pathogen and durgs. Thistechnology has several advantages over traditional immunoassays, such as simplicityof procedure, rapid operation and immediate results, low cost, no requirements fortechnical expertise or specialized equipment. In this study, a simple and rapidimmunochromatographic strip was developed for the detection of PCV2antibodies inswine. And several mAbs were screened and identificated. 1. Prokaryotic expression and characterization of Cap proteinThe ORF2gene, truncated nuclear localization signal (NLS) sequence, wasamplified by PCR from the recombinant plasmid pMD18T-ORF2. The purified PCRproduct was digested with BamHI and HindIII, and cloned into the pET28a vector.Then the recombinant plasmid was transformed to Escherichia coli (E. coli) BL21competent cells. The positive clone was grown at37℃in Luria-Bertani (LB)medium supplemented with100μg/mL kanamycin to the optical density of0.6at600nm, and then isopropyl-β-D-thiogalactopyranoside was added to a final concentrationof1mM. After8h of induction at30℃, cells were harvested by centrigugation(8000rpm for30min) and resuspended in20mL100mM Tris-HCl (pH8.0). Thecells were disrupted by ultra-sonication on ice and centrifuged at12000rpm for25min. The supernatant were collected and purified using nickel-nitrilotriacetic acid(Ni-NTA) resin following the manufacturer’s protocol. SDS-PAGE showed a newprotein band with a molecular weight of26KD. Western blot indicated that it couldreact specifically with PCV2positive sera. In ELISA, the protein was demonstrated tobe of high bioactivity. Thus, Cap protein successfully expressed in E. coli could beused as a diagnostic reagent and provides a basis for the study of subunit vaccine.2. Development and evaluation of an immunochromatographic strip for therapid detection of antibodies against Porcine circoviurs2A rapid (less than5min) immunochromatographic strip using gold-antigen probewas successfully developed and applied in the detection of porcine circovirus2(PCV2) antibodies in swine. Recombinant Cap protein truncated nuclear localizationsignal of PCV2was expressed and labeled with colloidal gold. This conjugate wasdispensed on conjugate pad as the detector. The staphylococcal protein A (SPA) andpurified PCV2antibodies (porcine origin) were blotted on the nitrocellulosemembrane for the test and control lines, respectively. Sensitivity and specificity of thestrip was evaluated using PCV2antisera as well as other antisera from pigs infectedwith different swine viruses.500clinical swine serum samples were detected both by strip and the commercial ELISA kit. The agreement of immunochromatographic stripand ELISA kit was94.00%. It showed that this strip possesses high sensitivity andspecificity and may be useful for clinical laboratories and rapid diagnosis in the field.3. Preparation and characterization of monoclonal antibody against PCV2Antibodies against PCV2were elicited by immunizing Balb/c mice with purifiedPCV2. MAbs were prepared routinely by cell fusion. The above-developed strip wasused to screen hybirdoma lines. Five positive hybridoma lines of3C9,6A5,8F9,9F4,15E4were screened out. Yet only6A5,8F9,9F4could react with PCV2. The striptiter for these hybridoma lines were1:4×105,1:3×105,1:4×105, respectively. ThesemAbs possess high-specificity, show on cross reaction with other proteins or virusesand lay the foundation for the development of strip test for the rapid detection ofPCV2. |
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