| PCV2, a single-stranded nonenveloped DNA virus with an unsegmented circular genome, is classified in the genus Circovirus of the family Circoviridae. The virus is the primary causative pathogen for several syndromes collectively known as porcine circovirus-associated disease (PCVAD). By now, PCV2infection is widespread in every pig-producing country in the world, and has been recognized one of global diseases in swine. More and more researchs focus on porcine circovirus associated diseases. PCV2infection was first detected in our country in2000. Since then, the rates of PCV2infection in pigs were kept highly. The molecular epidemiological survey suggests that PCV2were mutating constantly, and new genotypes were emerging. It is difficult for clinal diagnoses of PCVAD since PCV2usually coinfected with other pathogens. Thus, the molecular epidemiological surveillence of PCV2and development of detection method based on monoclonal antibody technology was very important for prevention and control of PCV2infection.1. Molecular epidemiological investigation of porcine circovirus type2from2012to2013in the partial area of Jiangsu provinceTo investigate the molecular epidemiology of Porcine circovirus type2(PCV2) in Jiangsu area, a total of139samples of suspected PCV2infection were collected and subjected to PCR detection and viral isolation. The results showed that the positive rate of PCR amplification was32.4%(45/139), and positive rate of viral isloation was23.7%(33/139). Thirty-three isolates of PCV2were further subjected to sequence, and the result of sequence analysis showed that the genotyps of33isolates all belonged to PCV2b, of which25isolates were grouped into PCV1C and8isolates were grouped into PCV1A/1B. These data indicate that PCV21C has been a dominant subgenotype in Jiangsu province.2. Prokaryotic expression of PCV2Cap geneThe capsid protein gene without nuclear localization signal (sCap) of PCV2was amplified by PCR, and cloned into4different vectors including pGEX-6p-1, pGEX-4T-1, pET-32a and ColdGX-6p-1, then transferred to E. coli BL21. The sCap proteins were expressed in the recombinant strain of pGEX-6p-1-sCap, pGEX-4T-1-sCap, pET-32a-sCap after induction. The sCap proteins could be expressed in soluable form in pGEX-6p-1-sCap by optimization of inducing conditions including the temperature, speed and IPTG concentration. The antiserum against Cap protein was prepared by immunization of mice with the purified sCap fusion protein by glutathione resin. The result of Indirect Fluorescence assay (IFA) revealed that the antisera produced a specific fluorescence in Dulac cells infected with PCV2, indicating that sCap protien possesses good antigenicity.3. Development of monoclonal antibodies against Cap protein of PCV2Balb/c mice were immunized with purified recombinant GST-sCap fusion protein, and spleen cells of immunized mouse were fused with myeloma cells (SP2/0) after forth immunization. Four monoclonal antibodies (McAbs) were screened by indirect ELISA, which using recombinant Cap protein as coated antigens and GST protein as negative control, these McAbs were named as1D6,5D11,6B7and6G5. The asites titers were1:64000,1:64000,1:128000and1:128000, respectively. The isotype of McAbs belonged to IgG2b, IgG1, IgG2a, IgGi, which were determined by the isotyping reagents. The result of IFA revealed that specific fluorescence was observed when Dulac cells were infected with PCV2and stained with McAbs, but no fluorescence was observed when cells were infected with PCV1, PRRSV or PRV. The result of Western blot confirmed that all four McAbs have a specific band with Cap protein, but not with GST protein. The result of neutralization test showed that only6B7mAb had neutralization titer.against PCV2. |
PDF Full Text Request