Abstract
In this research, the cocoonase gene was cloned by RT-PCR as an 860 bp fragment, including the signal peptide and thecore sequence of cocoonase gene. In order to investigate the function of signal peptide, recombinant transfer vectorpBacPAK8-Cocoonase-EGFP were constructed by fusing with enhanced green fluorescent protein (EGFP) gene toobserve under fluorescence microscope. The purified pBacPAK8-Cocoonase-EGFP DNA was co-transfected withlinear virus Bm-BacPAK6 DNA into BmN cells. The homologous recombination occurred in the cells and then therecombinant virus Bm-BacPAK8-Cocoonase-EGFP was obtained. BmN cell was infected with the recombinant virusBm-BacPAK8-Cocoonase-EGFP, and fluorescent signal was detected in most of the cells under fluorescencemicroscope at 72 hrs postinfection. Then BmN cells were harvested. Both SDS-PAGE and Western-blotting analysisindicated that the cocoonase was expressed successfully in silkworm (Bombyx mori) baculovirus expression vectorsystem. Furthermore, referred to Astrup methods,used fibrin plate process confirmed that expression product in vitrohad cellulolytic activity. We conclude that silkworm expression system can be used successfully to express functionalcocoonase.
Highlights
Cocoonase is a protease produced by silk moths during adult development and used for hydrolyzing and softening the end of the cocoon to permit exit of the adult moth
The plasmid pMD18-T/Cocoonase was digested with BamH I and Kpn I and ligated with pBacPAK8-enhanced green fluorescent protein (EGFP) which was digested with the same restriction enzymes to generate pBacPAK8-Cocoonase-EGFP
The cocoonase fragment could be isolated from the pBacPAK8-Cocoonase-EGFP vector after the recombinant plasmid was digested with BamH I and Kpn I (Fig. 3, lane 1)
Summary
Cocoonase is a protease produced by silk moths during adult development and used for hydrolyzing and softening the end of the cocoon to permit exit of the adult moth. Studies on the cocoonase have significance in the understanding of the physiological process of the silkmoth’s escape from cocoon, and in the exploitation of silk protein. The full-length sequence of the cocoonase gene has released in the National Center for Biotechnology Information (GenBank accession No EF428980) and the full-length cDNA of the cocoonase gene from Bombyx mori is 1047 bp with an ORF of 780 bp, 54 bp nucleotide sequence in 5ƍUTR (untranslated region), 210 bp nucleotide sequence in 3ƍUTR and termination codon TAA. The protein which was encoded by the cocoonase gene contains 260 amino acids. An alignment of the cDNA sequence with the silkworm genome sequences revealed that there were 4 introns and 5 exons within the open reading frame in the cocoonase gene from B. mori
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