Enhancement of recombinant peroxidase expression in baculovirus system

LUCIA ROMERO; ALEXANDRA TARGOVNIK; OSVALDO CASCONE,; MARÌA VICTORIA MIRANDA

Tucumàn

Congreso; SAIB; 2009

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 from the 5´end of polyhedrin gene (PPHS) in the 5´ 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 Sf9 cell line infected with MOI 1. In vivo assays were done on Spodoptera frugiperda and Rachiplusia nu larvae infected by intrahaemocoelical injection with 50 ml of 8.106 pfu.ml-1 of both viral suspensions. Although HRP level in vitro was the same with both viruses, enzyme levels achieved with AcMNPVHRP PPHS were 2 and 3  times higher than those reached with AcMNPVHRP. Thus, the PPHS introduction enhances secreted HRP expression level up to 132 and 298 mg . Kg-1 in S. frugiperda and R. nu respectively.