Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the β-galactosidase gene

• Published in: Archives of virology Vol. 146; no. 7; pp. 1355 - 1367
• Main Authors: RIBEIRO, B. M, GATTI, C. D. C, COSTA, M. H, MOSCARDI, F, MARUNIAK, J. E, POSSEE, R. D, ZANOTTO, P. M. A
• Format: Journal Article
• Language: English
• Published: Wien Springer 01.07.2001; New York, NY
• Subjects: Animals; Anticarsia gemmatalis; Anticarsia gemmatalis nucleopolyedrovirus; Bacteriology; Base Sequence; beta-Galactosidase - biosynthesis; beta-Galactosidase - chemistry; beta-Galactosidase - genetics; Biological and medical sciences; Cell Line; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Genetics; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Guanosine Triphosphate - genetics; Larva; Lepidoptera; Microbiology; Molecular Sequence Data; Mycology; Nucleopolyhedrovirus; Nucleopolyhedrovirus - genetics; Nucleopolyhedrovirus - pathogenicity; Occlusion Body Matrix Proteins; pol gene; polh gene; Promoter Regions, Genetic; Transfection; UFL-AG-286 cells; Viral Proteins - genetics; Viral Structural Proteins; Nucleopolyhedrovirus; Insecta; Escherichia coli; Primer; Galactosidase; Insertion; Baculovirus; Specificity; Baculoviridae; Gene; Bacteria; Infected cell; Vector; β-Galactosidase; Enterobacteriaceae; Enzyme; Transferases; Anticarsia gemmatalis; Virus; Polymerase chain reaction; Nucleotidyltransferases; Arthropoda; Glycosidases; DNA; Lepidoptera; Hydrolases; Isolate; Noctuidae; Invertebrata; O-Glycosidases; DNA-directed DNA polymerase
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s007050170096

We have constructed a transfer vector (pAgGal) containing the beta-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The beta-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay.