J Korean Soc Virol.  1997 Dec;27(2):239-256.

Recombination and Expression of VP1 Gene of Infectious Pancreatic Necrosis Virus DRT Strain in a Baculovirus,Hyphantria cunea Nuclear Polyhedrosis Virus

Abstract

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 peal-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 75 phage promoter and the 6x His region of the pQE-30 expression vector, and it was called pQEVP1. Again, the 6xHis-tagged VP1 DNA fragment in the pOEVPl was cleaved with EcoRl and transferred into the VP1 site of the pBacVPl, resulting pBacHis-VPl 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 6xHis-tagged VP1 protein produced by the pQEVPl 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 2.0x10(5) pfu/ml at 7 days postinfection.


MeSH Terms

Bacteriophages
Baculoviridae
Blotting, Western
Chromatography
Clone Cells
DNA
DNA, Complementary
Electrophoresis, Polyacrylamide Gel
Genome
Inclusion Bodies
Infectious pancreatic necrosis virus*
Molecular Weight
Nucleopolyhedrovirus*
Recombination, Genetic*
RNA
DNA
DNA, Complementary
RNA
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