Construction of Eukaryotic Expression Plasmid pcDNA3.1(+)-ORF1(P1)

To identify whether the porcine circovirus-like virus P1-ORF1 protein could be expressed in PK-15 cells, P1-ORF1 gene was amplified by polymerase chain reaction (PCR) and cloned into the pcDNA3.1(+)vector.The recombinant plasmid pcDNA3.1(+)-ORF1 was transfected into PK-15 cells by lipofectamine 2000, and the expression of ORF1 was identified on transcriptional and protein levels, respectively, using immunohistochemistry (IHC) and reverse transcription (RT-PCR). The results showed that the 359 bp target fragments was amplified by RT-PCR from total RNA extracted from the transfection cells and treated by recombinant DNase I.The recombinant plasmid pcDNA3.1(+)-ORF1 could work with polyclonal anti-P1-ORF1 specific reaction with IHC. pcDNA3.1(+)-ORF1 eukaryotic expression plasmid was constructed successfully, and it could be expressed in PK-15 cells, which contributed to the further study of the immunological functions of P1-ORF1 protein.