The Immungenicity Of Recombinant Plasmid Expressing The Major Nucleocapsid Gene Of Porcine Circovirus Type 2 And Epidemiological Investigation Of The Virus Infection In Local Regions

Porcine circovirus type 2 (PCV2) is the primary causative agent and a recently recognized pathogen of postweaning multisystemic wasting syndrome (PMWS) and other associated diseases, which have caused significant economic losses to the swine industry in many parts of the world. At present, there is no effective vaccine for the control and eradication of the disease, and seldom have any data on the epidemiological and serological investigation of the virus infection. In this study, a eukaryotic expression plasmid was constructed by introducing the major immunogenic gene from PCV2 into a eukaryotic expression plasmid vector, which was responsible for the development of PCV2 DNA vaccine. Furthermore, the humoral immune responses were dectected and analyzed in mice. Simultaneously, the investigation on PCV2 inapparently and persistently infection was also carried out, which laid a foundation for the study on the PCV2 pathogenesis and the prevention of the PCV2 infection.1. Construction of a eukaryotic recombinant plasmid expressing nucleocapsid protein of porcine circovirus type 2The nucleocapsid protein, also known as Cap protein is the dominant immunological agent and the only structural component of the viral capsid encoded by the ORF2 gene of PCV2. There are common antigenic determinants in Cap protein between the two types of PCVs, however, no cross-reaction of antigenicity is observed between them. Therefore, the ORF2 gene can be used to differentiate PCV1 and PCV2.The ORF2 gene of PCV2 was amplified by polymerase chain reacion (PCR) from the supernatant of PCV2 infected cells, and the amplified fragment was then choned into the eukaryotic expressing vector pcDNA3.1/V5-His-TOPO?. PCR, restriction endonuclease analysis and DNA sequencing were used to identify the recombinant plasmid. The results indicated that the ORF2 gene was successfully inserted into the vector. After that, PCV1-free Dulac cells were transfected with the recombinant plasmid named as pcDNA3.1/V5-His-orf2. The expression of Cap protein was confirmed by indirect immunofluorescence assay (IFA), and ORF2 gene in the pcDNA3.1/V5-His-orf2 could be expressed in vitro.2. The immunogenicity of the recombinant plasmid evaluated in miceBALB/c mice were vaccinated three times intramuscularly with the recombinant plasmid and serum samples collected from vaccinated and non-vaccinated mice at 7, 14, 21, 28, 35, 42 day post immunization were monitored for the antibody against PCV2 capsid protein by indirect ELISA (iELISA). The results showed that all vaccinated mice responded serologically by raising antibodies against PCV2 on 14 day post immunization, and the titers reached its highest level on 35 day post immunization, which suggested that the recombinant plasmid we constructed was of some immunogencity.3. Epidemiological investigation of porcine circovirus type 2 infection in local regions168 tissue samples and corresponding serum samples were collected in Nov, 2006 (high swine fever outbreak period) and Mar, 2007 from the slaughter house in Guangling District, Yangzhou. The presence of PCV2 and antibodies against PCV2 were dectected by PCR and iELISA separately. The results showed that the positive rates of antibodies against PCV2 were 91.36% and 100% for serum samples collected in 2006 and in 2007 respectively, while the positive rates of PCV2 were 58.02% and 47.13%, which suggested that PCV2 infection became widespread in swine, and about half of the swine were carried with PCV2 in the presence of elevated PCV2-specific antibodies. Moreover, the results also showed that the total levels of antibodies in 2006 were obviously higher than 2007, which revealed that persistent PCV2 infection could result in pigs with extremely high PCV2 antibody titres. This investigation also showed that there seemed no relationships between PCV2 carried and PCV2-specific antibody titres, which suggested that the antibody may be non-neutralizing once the infection was established, and PCV2-specific antibody in serum was not sufficient to prevent or clear the PCV2 virion in pigs.