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Engineering of an Oryctes Baculovirus Recombinant: Insertion of the Polyhedrin Gene from the Autographa californica Nuclear Polyhedrosis Virus

Allan M. Crawford

Entomology Division, Department of Scientific and Industrial Research, Private Bag, Auckland, New Zealand

A cotransfection method has been developed for the generation of recombinant Oryctes baculoviruses. The permissive coleopteran cell-line DSIR-HA-1179 was transfected with a mixture of Oryctes baculovirus DNA (strain PV505) and a transfer vector. The transfer vector, a pUC8-based plasmid, contained the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus, flanked by Oryctes baculovirus DNA from the HindIII fragment N. Oryctes baculovirus does not form plaques with DSIR-HA-1179 cells. Therefore an endpoint dilution method was used to screen for, recover and purify recombinants. There was no phenotypic character that could be used for detecting recombinants, so a dot blot assay was used to screen infected cultures for the presence of recombinants. A recombinant generated by this method contained the entire polyhedrin gene inserted at 98.03 map units from the designated start of the Oryctes baculovirus physical map. No evidence of transcription or translation of the polyhedrin gene was obtained.

Keywords: Oryctes baculovirus, cotransfection, NPV

Present address: MAFTech South Molecular Biology Unit, Biochemistry Department, University of Otago, P.O. Box 56, Dunedin, New Zealand.

Received 31 May 1988; accepted 19 December 1988.