| Porcine circovirus type 2 (PCV2) is now recognized as the causal agent of postweaning multisystemic wasting sysdrome(PMWS), an economically important wasting disease of postweaning pigs. PMWS was initially recognized in weaning piglets of a high-health- status Canadian herd in 1991, since then PMWS has become an economically important disease in virtually all regions of the world where pigs are produced. The most common clinical signs are unthriftiness wasting, dyspnea, enlarged lymphnodes, diarrhea, pallor, and jaundice.The most common diagnostic methods to detect PCV2 antibodies include indirect immunofluoresent assay (IFA) and immunoperoxidase monolayer assay (IPMA). IFA and IPMA require specific technical skills, additionally, examination of IFA and IPMA plates is tedious and time-consuming. Enzyme-linked immunosorbent assay (ELISA) may decrease the potential bias that may interfere with IFA or IPMA results. Recently many methods for detecting PCV2 antibodies using ELISA were reported, one of which was the indirect competitive ELISA (cELISA) with high sensitivity and specificity utilizing monoclonal antibodies to PCV2.Based on the published nucleotide sequence of ORF2 gene of porcine circovirus 2 strain DQ104422, a pair of primers were designed and synthesized. The ORF2 gene defecting the nuclear localization signal was amplified by polymerase chain reaction (PCR) from the supernatant of PCV2 infected cells, and the fragment mentiond above was cloned into pGEM-T easy vector. Restriction endonuclease analysis and DNA sequencing were used to identify the recombinant plasmid of pGEM-ORF2. The results indicated that the fragment were successfully inserted, and then the fragment was subcloned into the expression plasmid pGEX-6P-1. A recombinant vector was constructed and transformed into E. coLi BL21 cells. Then about 48kDa GST-Cap fusion protein was expressed in recombinant strain BL21(pGEX-ORF2) after induced by IPTG at 37℃, which was confirmed by SDS-PAGE and Western blotting analysis.An indirect ELISA assay coating with recombinant protein was established. The conditions for the ELISA were optimized as following: 3μg/mL as the final coating concentration of the recombinant protein, 1 hour at RT (about 25℃) and then overnight at 4℃for the recombinant protein coating, 1:200 as the dilution fold for serum samples. No cross-reations were observed between the antibody against PCV2 and the sera antibodies against classical swine fever (HCV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome (PRRSV). The ELISA established can be founded as antibody detection.kits for PCV2 infection.BALB/c mice was immunized subcutaneously with purified fusion protein. Mouse spleen cells were fused with SP2/0 myeloma cells after the last immunization. The fused cells were cultured continually with HAT medium. Hybridoma supernants were screened for the specific antibodies by indirect enzyme-linked immunosorbent assay. Two hybridoma cell strains against PCV2 Cap protein named 1D2 and 6D8 respectively were developed. The ELISA titers of culture supernatant were 1:256,1:1024, and those of ascites were 1:8192,1:131072 respectively. The results of ELISA indicated that the two monoclonal antibodies were only against PCV2 Cap protein but not reacted with pGEX-6P-1 protein,HCV,PPV and PRRSV. The results of IFA indicated that the 6D8 only reacted with PCV2 but not with PCV1, which seemed it to be specific to PCV2, while 1D2 can not react with PCV2 and PCV1 in IFA test. The analysis of Western-blot showed that 1D2 and 6D8 can react with the PCV2 Cap protein. The monoclonal antibodies could be used in the research of PCV2-ORF2 function and for discriminating the infection caused by PCV2.
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