Characterization of recombinant Autographa californica nuclear polyhedrosis virus (NPV) expressing the beta-galactosidase gene in both Sf21 and Bm5 cells by Bombyx mori NPV p143 helicase gene.
Genomic DNA of recombinant AcNPV expressing beta-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 beta-galactosidase either at the highest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) level were chosen for the characterization of beta-galactosidase expression in Bm5 and Sf21 cells. Expression level of beta-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 beta-galactosidase in Bm5 cells. However, expression of beta-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.
Jin
B R
BR
Laboratory of Insect Genetic Engineering, National Sericulture & Entomology Research Institute, Suwon, Korea.
Yoon
H J
HJ
Choudary
P V
PV
Kang
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology / Q000235:genetics; D002478:Cells, Cultured; D004265:DNA Helicases / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D018411:Spodoptera / Q000235:genetics; D014162:Transfection / Q000235:genetics; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics*
C091746:Ac-bro protein, Autographa californica nucleopolyhedrovirus; D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; D001616:beta-Galactosidase; D004265:DNA Helicases