Cloning And Expression Of The Cocoonase Gene From Bombyx Mori And Activity Analysis Of Its Promoter

Cocoonase is a protease secreted by silkmoth during its eclosion, and used for hydrolyzing and softening the cocoon end in convenient for the escape of silkmoth from the cocoon after eclosion. Studies on the silkmoth cocoonase are signality not only in the understanding of the physiological process of the silkmoth's escape from cocoon, but also in the exploitation of silk protein.Based on the partial sequence released in the National Center for Biotechnology Information ( GenBank accession No. AB257565 ), we used the rapid amplification of cDNA Ends method to clone the 5′-and 3′-end regions of the Cocoonase gene with the total RNA from the silkmoth head (Bombyx mori L.). The full-length cDNA of the Cocoonase gene from Bombyx mori is 1 047 bp with an ORF of 780 bp, 54 bp nucleotide sequence in 5′UTR ( untranslated region ), 210 bp nucleotide sequence in 3′UTR ( untranslated region ) and termination codon TAA ( GenBank accession No. EF428980 ). The protein which was encoded by the Cocoonase gene contains 260 amino acids.The protein molecular weight is about 27.6 kD and its isoelectric point is 8.89. 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 Bombyx mori, and all of them conformed the canonical GT-AG rules. Amino acid sequence analysing by Signal P 3.0 Server showed that there is a signal peptide of 22 amino acids at the N-termina1.Prediction of protein founctiona1 domain indicated that the trypsin-like serine protease domain of the cocoonase is from 34 to 254 amino acid. Then we got the full-length cDNA of the Cocoonase gene and analysed its control element by the biology software DNASTAR.The recombinant pET-32a-Cocoonase was constructed and introduced in E. coli BL21 to express a Trx-His-linked protein by IPTG. The result of SDS-PAGE demonstrated that the expression products migrated at a size of 48 kD. Furthermore, the Cocoonase gene was expressed using Bac-to-Bac baculovirus expression system. SDS-PAGE results showed that a specific band consistent with the expected size of fusion protein was detected around 27.6 kD. SEM micrographs were taken after treating silkworm silk with the expressed protein, and the results showed that the expressed protein can hydrolyze the sericine to some extent.To establish a bio-factory to produce foreign gene, the Cocoonase gene promoter was cloned and sequenced. A recombinant virus named Bac-CN-GFP-PolyA was constructed, in which the reporter gene GFP was driven by Cocoonase promoter. The promoter's activity was then characterized by recombinant virus expression assays in Sf9 cells of Spodoptera frugiperda. The results of a sequence analysis showed that the Cocoonase promoter possesses the characteristics of a eukaryotic promoter. Secondary structure analysis indicated that the sequence of the promoter region forms a complicated stem-loop structure. This may relate to the tissue speciality or the timing and activity of the protein expression. The result of virus experimentation showed that the Cocoonase gene promoter could drive the report gene expressing in the Sf9 cells.