Construction of goose parvovirus VP3 gene eukaryotic expression vector and expression in Vero cell line.

A pair of primers was designed and synthesized according to nucleotide sequence of goose parvovirus(GPV) B strain and VP3 gene was amplified from the DNA of goose parvovirus isolated in Jilin province,China.The PCR amplified VP3 gene was cloned into pMD18-T vector.VP3 gene was sequenced and cloned into the eukaryotic expression vector pVAX1.The recombinant plasmid was used to transform competent Escherichia coli DH5α and the positive clones were identified by PCR and restriction enzyme digestion.Then the recombinant pVAX1-VP3 was transfected to Vero cell line with LipofectamineTM2000.The expression of pVAX1-VP3 was detected by RT-PCR and indirect immunofluorescence test.The result showed that a 1 605 bp VP3 gene fragment was amplified from the GPV DNA and was 96.2% homology with the sequence of GPV B strain.PCR and restriction enzyme digestion confirmed that VP3 gene was inserted into the eukaryotic expression vector pVAX1.The 1 000-2 000 bp DNA fragment was amplified from the RNA extracted from the Vero cell line transfected with pVAX1-VP3 by the RT-PCR method and the VP3 specific protein on the cell was detected by indirect immunofluorescence test.