Construction Of PCV2 Infectious Clone And Research Of The Function Of ORF3 Protein

Porcine circovirus(PCV) was divided into the family porcine circovirus, genus porcine circovirus, which included two serotypes of PCV1 and PCV2. PCV1 has no pathogenicity of swine, but PCV2 is the main pathogen of postweaning multisystemic wasting syndrome (PMWS) and is closely related to many diseases. PCV2 has induced great economic loss in pig industry, so PCV2 has received much attention. Recently, a theory has been put forward and confirmed that ORF3 protein had effect on apoptosis and was relationship to pathogenicity, researchers has paid more attention to the function of this protein. Based on the existing theory, we first planed to construct infectious clone of PCV2, and then change the ORF3 protein by site directed mutagenesis, at last we research the effects of ORF3 protein on cell proliferations and apoptosis through experiments. We researched the function of ORF3 protein at the whole virus level and planned to determine which part of the ORF3 protein plays the leading role on its apoptosis function. This study included three parts:Partâ… : Cloning and sequencing of complete genome of PCV2-SD strain.One strain of PCV2 (named PCV2-SD)was isolated from pigs which suspected infected with PMWS. The virus genome was got by PCR and sequence analysis showed that the homology of nucleotide sequence between PCV2-SD and reference sequence on GeneBank was 96%-98.9%,the homology of ORF3 amino acid is 92%-99%.Partâ…¡: Construction of PCV2-SD Strain infectious clone and the mutation clone.Two virus genome were connected subsequently into pUC19 vector to construct a double copy infectious clone. Without affecting the normal amino acid sequence of ORF1, ORF3 was transformed using PCR site-directed mutagenesis techniques. Four ORF3 deleted infectious clones separately named pUMu1-H1H2, pUMu27-H1H2, pUMu51-H1H2 and pUMu79-H1H2 were constructed by ending expression at position No. 1,27,51,79 of ORF3 amino acid. These five infectious clones were transfected into PK-15 cells, and identified by IFA. The results showed that the five clones could save infectious virus particles. Partâ…¢: Research of the effect of ORF3 protein on the biological properties of PCV2-SD.Five isolates of virus which were transfected into PK-15 cells were equivalent to inoculate into PK-15 cells. Virus genome was respectively extracted at 1h, 12h, 24h, 36h and 48h after inoculation. Virus genome quantification was done by quantitative PCR. The results show that the complete or partial deletion of ORF3 had no effect on infection of PCV2 on PK-15 and on the ability of proliferation of PCV2 in PK-15. PK-15 cells were collected at 48h and its apoptosis were detected. The results showed the PCV2 without ORF3 protine still have apoptosis to PK-15, ORF3 is not the only pathogenic factor. Function of cell apoptosis of virus is not obvious when the ORF3 protein is complete deleted or ORF3 length is less than 27 amino acids, and the apoptosis is becoming significantly as the ORF3 protein becoming longer . In this research, the primary acting site of ORF3 was initially targeting at the 27-104 amino acids.