Porcine Parvovirus: PCR Detection, Isolation, Identification, And Its Monoclonal Antibody Development

Porcine Parvovirus(PPV) is one of major causative agents, resulted in a sydrome of reproductive failure in swine, which includes stillbirths, mummified failure in early embryonic death, and infertility. There is no obvious sydromes in pregnant sows. Porcine parvovirus infection becomes a world-wide problem and causes a great economic loss in pig industry in China. At present, a lot of dignostic methods for porcine parvovirus infection have been developed based on its aetiology and serology.Based on the gene sequences of porcine parvovirus in GenBank, a pair of primers were designed and synthetized. Expected fragments is amplified by PCR successfully, which was about 500bp. 120 clinical samples were investigated using with this technique. As a result, two PPV strains (No.1 and No.3) were isolated by cell culture. Physical and chemical characterisitics were identified for one of them, and it resisted acid (pH3.0), trypsin, chloroform, ether and heat(56℃), its TCID50 was 0.1ml 10-3.85.The BALB/c mice were immunized subcutaneously with PPV, which was purified with polyethylene glycol. After being immunized three times, the mice were immunized intraveinally with equal the dose for the final immunization. Three days later, spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (iELISA) was used to screen hybridoma cells, and limiting dilution method was applied to subclone positive hybridoma cells three times, and two positive clones, designated as 4B2 and 4G6 respectively, were obtained from hybridoma cell lines. The titers of their ascitic fluids were 10×210,10×27 in ELISA, respectively. Both of them were subtyped to be IgM. 4B2 reacted with PPV specifically in indirect immunofluorecent assay (IFA) .