Characterization of Recombinant Autographa californica Nuclear Polyhedrosis Virus (NPV) Expressing the β-Galactosidase Gene in both Sf21 and Bm5 Cells by Bombyx mori NPV p143 Helicase Gene

Byung Rae Jin, Hyung Joo Yoon, Prabhakara V Choudary, Seok Kwon Kang

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Genomic DNA of recombinant AcNPV expressing β-galactosidase was cotransfected with p143 helicase gene of BmNPV into Sf21 cells. Ac-Bm hybrid viruses capable of replicating in both Bm5 and Sf21 cells were isolated. Ac-Bm hybrid viruses expressing β-galactosidase either at the highest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) level were chosen for the characterization of β-galactosidase expression in Bm5 and Sf21 cells. Expression level of β-galactosidase and replication of Ac-Bm hybrid virus-HE in Sf21 cells were nearly identical to those of recombinant AcNPV. Furthermore, replication of Ac-Bm hybrid virus-HE in Bm5 cells was similar to that of wild-type BmNPV, and Ac-Bm hybrid virus-HE clearly expressed β-galactosidase in Bm5 cells. However, expression of β-galactosidase by Ac-Bm hybrid virus-HE in Bm5 cells was significantly lower than that expressed in Sf21 cells. The titer of Ac-Bm hybrid virus-HE determined by plaque assays in Bm5 cells was similar to that determined in Sf21 cells, but the plaque size formed by Ac-Bm hybrid virus-HE in Bm5 cells was apparently smaller than that formed in Sf21 cells. In addition, expression levels and virus titers of Ac-Bm hybrid virus-LE in Sf21 and Bm5 were significantly lower than those of Ac-Bm hybrid virus-HE. Therefore, DNA sequences were determined for the region of the p143 gene controlling the host range in Ac-Bm hybrid viruses. The results showed that the deduced amino acid sequences of Ac-Bm hybrid virus-HE were almost identical to those of BmNPV. There were differences only in amino acids at positions 461 and 470, whereas those of Ac-Bm hybrid virus-LE were different at position 461, 470, 514, and 528 from those of BmNPV. In conclusion, our results clearly demonstrated that Ac-Bm hybrid virus-HE has an additional advantage of expanded host range for producing recombinant proteins.

Original languageEnglish (US)
Pages (from-to)762-768
Number of pages7
JournalMolecules and Cells
Volume7
Issue number6
StatePublished - Dec 31 1997

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Galactosidases
Nucleopolyhedrovirus
Sf9 Cells
Bombyx
Viruses
Genes
Host Specificity
Recombinant DNA

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Characterization of Recombinant Autographa californica Nuclear Polyhedrosis Virus (NPV) Expressing the β-Galactosidase Gene in both Sf21 and Bm5 Cells by Bombyx mori NPV p143 Helicase Gene. / Jin, Byung Rae; Yoon, Hyung Joo; Choudary, Prabhakara V; Kang, Seok Kwon.

In: Molecules and Cells, Vol. 7, No. 6, 31.12.1997, p. 762-768.

Research output: Contribution to journalArticle

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abstract = "Genomic DNA of recombinant AcNPV expressing β-galactosidase was cotransfected with p143 helicase gene of BmNPV into Sf21 cells. Ac-Bm hybrid viruses capable of replicating in both Bm5 and Sf21 cells were isolated. Ac-Bm hybrid viruses expressing β-galactosidase either at the highest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) level were chosen for the characterization of β-galactosidase expression in Bm5 and Sf21 cells. Expression level of β-galactosidase and replication of Ac-Bm hybrid virus-HE in Sf21 cells were nearly identical to those of recombinant AcNPV. Furthermore, replication of Ac-Bm hybrid virus-HE in Bm5 cells was similar to that of wild-type BmNPV, and Ac-Bm hybrid virus-HE clearly expressed β-galactosidase in Bm5 cells. However, expression of β-galactosidase by Ac-Bm hybrid virus-HE in Bm5 cells was significantly lower than that expressed in Sf21 cells. The titer of Ac-Bm hybrid virus-HE determined by plaque assays in Bm5 cells was similar to that determined in Sf21 cells, but the plaque size formed by Ac-Bm hybrid virus-HE in Bm5 cells was apparently smaller than that formed in Sf21 cells. In addition, expression levels and virus titers of Ac-Bm hybrid virus-LE in Sf21 and Bm5 were significantly lower than those of Ac-Bm hybrid virus-HE. Therefore, DNA sequences were determined for the region of the p143 gene controlling the host range in Ac-Bm hybrid viruses. The results showed that the deduced amino acid sequences of Ac-Bm hybrid virus-HE were almost identical to those of BmNPV. There were differences only in amino acids at positions 461 and 470, whereas those of Ac-Bm hybrid virus-LE were different at position 461, 470, 514, and 528 from those of BmNPV. In conclusion, our results clearly demonstrated that Ac-Bm hybrid virus-HE has an additional advantage of expanded host range for producing recombinant proteins.",
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