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KMID : 0578319970070060762
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Molecules and Cells
1997 Volume.7 No. 6 p.762 ~ p.768
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Characterization of Recombinant autographa califronia Nuclear Polyhedrosis Virus (NPV) Expressing the β-Galactosidase Gene in both Sf21 and Bm5 Cells by Bombyx mori NPV p143 helicase Gene
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Jin, Byung Rae
Yoon, Hyung Joo/Choudary, Prabhakara V./Kang, Seok Kwon
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Abstract
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Genomic DNA of recombinant AcNPV expressing β-galactosidase was cotransfected with p143 helicase gene of BmNPV into Sf21 cells. Ac-Bm hygrid viruses capable of replicating in both Bm5 and Sf21 cells were isolated. Ac-Bm hybrid viruses expressing β-galactosidase either at the hightest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) elvel 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 virusp-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 assay 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 were apparently smaller than formed in Sf21 cells. In addition, expression levels and virus titers of Ac-Bm hybrid virus-LE in Sf21 and Bm4 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 amono 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 demostrated that Ac-Bm hybrid virus-HE has an additional advantage of expanded host range for producing recombinant proteins.
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KEYWORD
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