1887

Abstract

A bacterial gene was inserted into an isolate of the nuclear polyhedrosis virus (LdMNPV). The transfer vector was constructed by site-directed mutagenesis of the translation start site of the LdMNPV polyhedrin gene, within the II E fragment of the viral genome. A multiple cloning sequence was inserted at this start site and used for the insertion of the gene into the transfer plasmid. Liposome transfection was used to cotransfect tissue culture cells with viral DNA and the transfer plasmid. Recombinant LdMNPV isolates were purified by isolation of plaques producing β-galactosidase but not polyhedra. Restriction enzyme fragment profiles were used to determine the site of the gene insertion, and DNA sequencing of the 5′ and 3′ ends of the gene insert and the adjoining polyhedrin promoter and coding regions was performed to identify its precise location. Expression of the gene was examined by studying virus-induced protein using [S]methionine pulse-labelling, SDS-PAGE fractionation and autoradiography. Expression of β-galactosidase was examined in tissue culture cells using colorimetric assays. The maximum rate of β-galactosidase production was approximately 50 international units (IU)/10 tissue culture cells/day between 3 and 4 days post-infection (p.i), and the peak total expression was 158 IU/10 cells 5 days p.i. β-Galactosidase activity was first detected 48 h p.i. in haemolymph samples from fourth instar larvae injected with 10 p.f.u. of virus. The peak β-galactosidase activity in larval haemolymph samples was 1931 IU/ml of haemolymph at 11 days p.i., just prior to death.

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1992-06-01
2022-12-06
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