CHARACTERIZATION OF RECOMBINANT AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS (NPV) EXPRESSING THE BETA-GALACTOSIDASE GENE IN BOTH SF21AND BM5 CELLS BY BOMBYX-MORI NPV P143 HELICASE GENE

Genomic DNA of recombinant AcNPV expressing beta-galactosidase was cot ransfected with p143 helicase gene of BmNPV into Sal cells, Ac-Bm hybr id viruses capable of replicating in both Bm5 and Sf21 cells were isol ated. Ac-Bm hybrid viruses expressing beta-galactosidase either at the highest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) lev el were chosen for the characterization of beta-galactosidase expressi on in Bm5 and Sf21 cells. Expression level of beta-galactosidase and r eplication of Ac-Bm hybrid virus-HE in Sf21 cells were nearly identica l to those of recombinant AcNPV. Furthermore, replication of Ac-Bm hyb rid 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 cell s. 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 Bm 5 cells was similar to that determined in Sf21 cells, but the plaque s ize formed by Ac-Bm hybrid virus-HE in Bm5 cells was apparently smalle r than that formed in Sf21 cells. In addition, expression levels and v irus titers of Ac-Bm hybrid virus-LE in Sal and Bm5 were significantly lower than those of Ac-Bm hybrid virus-HE, Therefore, DNA sequences w ere determined for the region of the p143 gene controlling the host ra nge in Ac-Bm hybrid viruses. The results showed that the deduced amino acid sequences of Ac-Bm hybrid virus-HE were almost identical to thos e of BmNPV. There were differences only in amino acids at positions 46 1 and 470, whereas those of Ac-Bm hybrid virus-LE were different at po sition 461, 470, 514, and 528 from those of BmNPV. In conclusion, our results clearly demonstrated that Ac-Bm hybrid virus-HE has an additio nal advantage of expanded host range for producing recombinant protein s.