MIRANDA Maria Victoria
congresos y reuniones científicas
Development of recombinant baculovirus for peroxidase production in orally-infected insect larvae
ROMERO, L.; LEVIN, G.; LOUSTAU, M.; TABOGA, O.; CASCONE, O.; MIRANDA, M.V.
Congreso; XLII Reunión anual SAIB; 2006
.... For recombinant protein production, Rachiplusia nu (R. nu) larvae can be infected with Autographa californica virus (AcMNPV) by injection or orally. Oral infection is less laborious and time consuming. Most recombinant baculovirus are constructed by replacing the polyhedrin gene with the foreign gene but, as these viruses do not produce polyhedra (occ-), they are not able to efficiently infect larvae orally. In this study we assess two different approaches to produce recombinant baculovirus for oral infection. 1) Mixed polyhedra obtention by co-infection of wild type AcMNPV and AcMNPV HRP+/occ-: A Sf9 subconfluent monolayer was inoculated with both viral populations at a different multiplicity of infection and various proportions. 2) AcMNPV HRP+/occ+ construction: HRP gene was cloned under the polyhedrin promoter, and polyhedrin gene under p10 promoter. A similar monolayer was co-transfected with transfer vector pBacPack-HRP and bAcGOZA. Fourth instar R. nu larvae were starved for 6 h and then they were fed with artificial diet contaminated with 1x106 pol/100 mg of both polyhedra suspensions. At 4º day post-infection hemolymph was collected and peroxidase activity was measured. Polyhedra baculoviruses obtained by both strategies resulted in successful HRP production in orally- infected insect larvae.