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

ROMERO, LUCÍA VIRGINIA; TARGOVNIK, ALEXANDRA MARISA; TABOGA, OSCAR; CASCONE, OSVALDO; 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. Polyhedra were characterized in both cases and they 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.