• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.264-269
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• 1994

To determine the nucleotide sequence of the ds RNA segment B containing the RNA dependent RNA polymerase (RdRp) gene of the DRT strain of infectious pancreatic necrosis virus (lPNV), the cDNA of the ds RNA segment B of the DRT strain of IPNV was synthesized using the reverse transcriptase (RT)-polymerase chain reaction (PCR) and its cDNA nucleotide sequence was determined. The DRT segment B was 2, 783 bp long and contained only a single long open reading frame (ORF) of 2, 535 bp in length. This ORF nucleotides encoded the VPl protein, the putative RdRp of IPNV. The VPl protein comsisted of 845 amino acids. The molecular weight of the RdRp, as deduced from the nucleotide sequence, is 94, 426. The nucleotide sequence of the ORF of the DRT showed 89.7% homology to the Jasper strain, but 80.8% to the Sp strain. The amino acid sequence of the ORF of the DRT sho.wed 97.6% homology to the Jasper strain, but 88.7% to the Sp strain. The conserved GTP-binding motif was detected in VPl protein.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.