MIRANDA Maria Victoria
congresos y reuniones científicas
Enhancement of Recombinant Peroxidase Expression in the Baculovirus System
LUCIA ROMERO; TARGOVNIK A; CASCONE O; MIRANDA MARÍA VICTORIA
Congreso; IBS 2010. 14th International Biotechnology Symposium and Exhibition. Biotechnology for the Sustainability of Human Society.; 2010
Baculovirus-insect cell system is a popular choice for heterologous gene expression when a eukaryotic environment is required. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is by far the most widely used baculovirus expression vector. Many attempts were done in order to increase recombinant protein expression levels. In this work a new recombinant virus was constructed with the aim of enhancing horseradish peroxidase (HRP) expression level by introducing a 15 pb element (PPHS) from the 5´end of the polyhedrin gene in the 5´ end of the peptide-signal GP67-HRP fusion protein gene. HRP follows the secretion route. This virus (AcMNPVHRP PPHS) was compared with a lacking-PPHS one (AcMNPVHRP) for HRP expression. In vitro experiments were performed in the Sf9 cell line, infected with a MOI 1. In vivo assays were done on Spodoptera frugiperda and Rachiplusia nu larvae infected by intrahaemocoelical injection with 50 ml of 7.106 pfu.ml-1 of each viral suspension and larvae were reared at 24º C. HRP expression level in vitro was similar with both viruses. In contrast, PPHS introduction enhanced HRP expression level from 55.4 and 104.8 to 126 and 297 mg.Kg-1 larvae (2.3 and 2.8 times) for S. frugiperda and R. nu, respectively. In addition to PPHS introduction, temperature had a strong effect on HRP expression: when the larvae were reared at 27° C, HRP expression level was 314.6 and 480.1 mg HRP/Kg-1 larvae (2.5 and 1.6 times higher than when reared at 24° C) for S. frugiperda and R. nu respectively. Summarizing, in this system the combined effect of PPHS and temperature rise to 27° C resulted in an increase of HRP expression of 5.7 and 4.6 times for S. frugiperda and R. nu respectively.