Jin B R, Yoon H J, Choudary P V, Kang S K
Laboratory of Insect Genetic Engineering, National Sericulture & Entomology Research Institute, Suwon, Korea.
Mol Cells. 1997 Dec 31;7(6):762-8.
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.
将表达β-半乳糖苷酶的重组AcNPV的基因组DNA与BmNPV的p143解旋酶基因共转染到Sf21细胞中。分离出能够在Bm5和Sf21细胞中复制的Ac-Bm杂交病毒。选择在最高（Ac-Bm杂交病毒-HE）或最低（Ac-Bm杂交病毒-LE）水平表达β-半乳糖苷酶的Ac-Bm杂交病毒，用于表征Bm5和Sf21细胞中β-半乳糖苷酶的表达。Ac-Bm杂交病毒-HE在Sf21细胞中β-半乳糖苷酶的表达水平和复制情况与重组AcNPV几乎相同。此外，Ac-Bm杂交病毒-HE在Bm5细胞中的复制情况与野生型BmNPV相似，并且Ac-Bm杂交病毒-HE在Bm5细胞中明显表达β-半乳糖苷酶。然而，Ac-Bm杂交病毒-HE在Bm5细胞中β-半乳糖苷酶的表达明显低于在Sf21细胞中的表达。通过噬斑测定法在Bm5细胞中测定的Ac-Bm杂交病毒-HE的滴度与在Sf21细胞中测定的滴度相似，但Ac-Bm杂交病毒-HE在Bm5细胞中形成的噬斑大小明显小于在Sf21细胞中形成的噬斑大小。此外，Ac-Bm杂交病毒-LE在Sf21和Bm5中的表达水平和病毒滴度明显低于Ac-Bm杂交病毒-HE。因此，测定了Ac-Bm杂交病毒中控制宿主范围的p143基因区域的DNA序列。结果表明，Ac-Bm杂交病毒-HE推导的氨基酸序列与BmNPV的几乎相同。仅在第461和470位氨基酸存在差异，而Ac-Bm杂交病毒-LE在第461、470、514和528位氨基酸与BmNPV的不同。总之，我们的结果清楚地表明，Ac-Bm杂交病毒-HE在生产重组蛋白方面具有宿主范围扩大的额外优势。
