Expression of an eclosion hormone gene in insect cells using baculovirus vectors

Abstract

The ability of a baculovirus vector system to produce insect eclosion hormone (EH) was investigated by insertion of a Manduca sexta cDNA encoding EH into the genome of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) such that transcription was under the control of a strong, modified polyhedrin promoter. Two polypeptides, approx. 6.0–6.5 kDa, were synthesized in and secreted from recombinant virus-infected cells but not wild-type (wt) virus-infected cells. Both polypeptides were immunoprecipitated by polyclonal antiserum directed againnst natural M. sexta EH. Immunoblotting revealed only a single polypeptide, suggesting that only one form is stable in the expression system. The size of this polypeptide and its elution from a reverse phase HPLC column indicate that this polypeptide is similar, if not identical, in structure to natural EH. Bioassay data revealed that biologically active EH was synthesized and secreted at high levels (e.g. 10 μg of active polypeptide per 10 6 cells). Thus, the baculovirus expression system is an excellent source of EH for further studies of this unique insect neurohormone.