Sequence Analysis Of NS1 And VP1 Genes Of GPV CZ Strain And Expression Of NS1 Gene

According to the sequences of goose parvovirus (GPV) B strain genome nucleotide published in genbank, a pair of specific primers was designed. NS1 and VP1 genes of GPV CZ strain were amplified by polymerase chain reaction (PCR) and PCR products were cloned into pcDNA3.1 vector. The two fragments were sequenced and analyzed with reference GPV strains and Muscovy duck parvovirus (MDPV) strains. The results showed that the identity of NS1 and VP1 gene between CZ and other strains isolated in China is higher than CZ and B that isolated in Hungary. This high identity between GPV CZ and MDPV indicated that they would be come from the same ancestor.The fragment of NS1 was subcloned into the prokaryotic expression vector pGEX-6p-1, and transformed into the competent cells of BL21 (DE3) E. coli. After 4h incubation, expression of the NS1 protein was induced by 0.05mmoL IPTG. After brief sonication and centrifugation, the supernatant of the cell lysate was minced for mouse immunization. Seven days after the final immunization, the serum was collected for immunofluorescence assay (IFA) with goose embryo firoblast (GEF) cells infected with GPV CZ strains. IFA and Western-blot results showed that the product of NS1 was a good immugen.The fragment of NS1 was subcloned into baclovirus tranfer vector pFastBac1. The transfer vector pFastBac1-NS1 was transformed into DH10Bac E.coli cells by site-specific transposition. NS1 gene was integrated into Bacmid and recombinant shuttle vector named Bacmid-NS1. The recombinant baculovirus, designated as reBac-NS1was obtained from Sf9 cells transfected with shuttle vector Bacmid-NS1 mediated by lipofectin. rNS1 was expressed in the Sf9 cells at 96h post-infection with reBac-NS1, which was identified by IFA and Western blot.