Construction and Characterization of Novel Baculovirus Expression Vector System Using Bombyx mori Nucleopolyhedrovirus

Abstract

A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, an extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBm bacmid could be replicated only in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared to that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established. Also, an easy, fast and mass purification system was developed using baculovirus expression vector system (BEVS) in which Expression by translational fusion of the polyhedrin-enhanced green fluorescence protein (EGFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Cry1Ac foreign protein fused with the partial polyhedrin and EGFP gene at the C-terminus, including an enterokinase (EK) site between EGFP and Cry1Ac protein, was expressed in insect cells. Cells infected by BmPolh19EG-1Ac or BmPolh32EG-1Ac produced fluorescent granules. The Cry1Ac fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, Cry1Ac presented mainly in the supernatant, and this supernatant fraction also showed a pure Cry1Ac band. These results suggest that the combined procedure of polyhedrin fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.