Expression of Cocoonase in Silkworm (Bombyx mori) Cells by Using a Recombinant Baculovirus and Its Bioactivity Assay

Abstract

In this research, the cocoonase gene was cloned by RT-PCR as an 860 bp fragment, including the signal peptide and the core sequence of cocoonase gene. In order to investigate the function of signal peptide, recombinant transfer vector pBacPAK8-Cocoonase-EGFP were constructed by fusing with enhanced green fluorescent protein (EGFP) gene to observe under fluorescence microscope. The purified pBacPAK8-Cocoonase-EGFP DNA was co-transfected with linear virus Bm-BacPAK6 DNA into BmN cells. The homologous recombination occurred in the cells and then the recombinant virus Bm-BacPAK8-Cocoonase-EGFP was obtained. BmN cell was infected with the recombinant virus Bm-BacPAK8-Cocoonase-EGFP, and fluorescent signal was detected in most of the cells under fluorescence microscope at 72 hrs postinfection. Then BmN cells were harvested. Both SDS-PAGE and Western-blotting analysis indicated that the cocoonase was expressed successfully in silkworm (Bombyx mori) baculovirus expression vector system. Furthermore, referred to Astrup methods,used fibrin plate process confirmed that expression product in vitro had cellulolytic activity. We conclude that silkworm expression system can be used successfully to express functional cocoonase.