Molecular characterization of polyhedra to select the best recombinant baculovirus for protein expression in orally infected insect larvae.

ROMERO L.; ALEXANDRA TARGOVNIK; OSCAR TABOGA; OSVALDO CASCONE; MIRANDA MARÍA VICTORIA

Current Topics in Virology

Research Trends

Año: 2010 vol. 8 p. 49 - 57

0972 4591

Two recombinant baculoviruses were compared as expression vectors in orally infected Rachiplusia nu larvae using horseradish peroxidase isozyme C (HRP) as the model protein. The strategies assessed for polyhedra generation were mixed polyhedra and recombinant polyhedrin-plus baculovirus (polh+ AcMNHRP) production. Recombinant polyhedra production by infected insect cell cultures was studied in both cases and the polyhedra obtained were used as inocula in oral infection assays using R. nu larvae through diet contamination. At 4 days post-infection, mixed polyhedra obtained at multiplicities of infection of wild type:recombinant virus ratios of 2:10 and 2:20 reached the highest HRP levels in hemolymph: 78.9 ± 9.9 U ml-1 and 37.1 ± 20.4 U ml-1 respectively. On the other hand, enzyme activity was significantly higher with polh+ AcMNHRP polyhedra: 203.6 ± 27.1 U ml-1. According to real time PCR, the recombinant virus never exceeded 20% of the total virus population within mixed polyhedra, in agreement with the lower HRP expression levels achieved. In conclusion, real time PCR is a rapid effective method for characterizing polyhedra composition and for selecting the best viral expression vector. We also proved that the performance of polh+ AcMNHRP and polh- AcMNHRP as HRP expression vectors was the same using either infected insect cell cultures or larvae, indicating that there was no interference between polyhedrin and HRP expression or a different fitness between both viral types. Oral infection of susceptible larvae is an economical and practical system to produce an active enzyme. In a typical experiment, maximal yield of HRP corresponds approximately to 23 µg per infected larvae.