MIRANDA Maria Victoria
congresos y reuniones científicas
Performance comparison of two recombinant baculovirus vectors for high-level expresión of peroxidase in insect larvae.
ROMERO L.; LEVIN G.; TARGOVNIK A.; LOUSTAU M.; SEGURA M.; WOLMAN F.; TABOGA O.; MIRANDA MV.; CASCONE O.
Congreso; 13 th European Congress on Biotechnology; 2007
Horseradish peroxidase isozyme C (HRPC) is an important commercial biocatalyst. This enzyme is a highly glycosylated haemoprotein stabilized by four disulfide bonds. These structural properties complicate its recombinant expression in prokaryotes. Despite the bacteria and yeast systems are preferred, they often fail to express eukaryotic proteins in active form. On the other hand, the baculovirus insect cell system is a popular choice for expressing foreign genes. Recently, we have expressed and purified HRPC as a fusion protein in Spodoptera frugiperda cell lines (Segura et al, 2005; Levin et al, 2005. Nevertheless, insect larvae could be used like factories to scale up the enzyme production because of their low cost in comparison with cell cultures. Rachiplusia nu and S. frugiperda larvae are permissive hosts for infection with Autographa californica nucleopolyhedrosis virus (AcMNPV) either by orally inoculated polyhedra or by injection of budded viral particles directly into haemolymph. Oral infection is the preferred methodology when a high number of insects must be inoculated. However, the most of recombinant baculovirus is constructed conventionally by replacing the polyhedrin gene with the foreign gene of interest.These type of recombinant baculovirus are known as phenotype occ- due to the incapability to produce polyhedra and its low efficiency to infect larvae orally. The aim of this study was the construction of a recombinant baculovirus occ+ as a vector to produce high-level expression of HRPC in insect larvae by oral infection. Two strategies were assayed; the first one was mixed polyhedra production by co-infection of insect cell lines with wild type AcMNPV (1.9x108 pfu/ml) and AcMNPV HRP+/occ- (2.4x108 pfu/ml). Subconfluent monolayers of Sf9 (7.105 cell/well) were infected at different multiplicity of infection (MOI) of both viral populations and relationships between them. After 4 days post-infection (dpi) the cells were collected and mixed polyhedra were purified. The second strategy was the construction of a recombinant baculovirus AcMNPV HRP+/occ+ in which HRP gene was cloned under the polyhedrin promoter and polyhedrin gene under p10 promoter. Four instar larvae of R. nu and S.frugiperda were starved for 24 hours and then, they were fed with artificial diet contaminated with 1x106 pol/100 mg of both polyhedra suspensions. At 4 dpi and 8 dpi respectively, the haemolymph was collected and peroxidase activity was measured. The best condition for R. nu larvae infection with mixed polyhedra was with MOI ratio 2:10 (wild type virus: recombinant virus), with a HRPC activity of 78,5 U/ml (31,1 mg/l). However, the enzyme activity in the haemolymph of R. nu infected with AcMNPV HRP+/occ+ polyhedra was approximately 50% higher (118,5 U/ml, 46.8 mg/l). When S. frugiperda larvae were infected with AcMNPV HRP+/occ+ polyhedra, HRP activity was higher than that obtained with mixed polyhedra but 137 times lower than that achieved in R. nu larvae. According with these results, R. nu larva is the best choice for HRPC production by oral infection with AcMNPV HRP+/occ+. This strategy results more efficient and practical than the production of mixed polyhedra. The method described is an alternative - based on recombinant technology - to obtain high mass production of an important biocatalyst.