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http://dx.doi.org/10.7852/jses.2014.52.1.45

Cloning and functional expression of a cecropin-A gene from the Japanese oak silkworm, Antheraea yamamai  

Kim, Seong-Ryul (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
Choi, Kwang-Ho (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
Kim, Sung-Wan (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
Goo, Tae-Won (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
Hwang, Jae-Sam (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
Publication Information
Journal of Sericultural and Entomological Science / v.52, no.1, 2014 , pp. 45-51 More about this Journal
Abstract
A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.
Keywords
Antheraea yamamai; Cecropin; KSI-CecA; Antibacterial activity;
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