Journal of Sericultural and Entomological Science (한국잠사곤충학회지)

Korean Society of Sericultural Science (한국잠사학회)
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Isolation and purification of a cecropin-like antimicrobial peptide from the japanese oak silkworm, Antheraea yamamai

천잠 세크로핀 항균펩타이드 분리 및 정제

  • Kim, Seong-Ryul (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Goo, Tae-Won (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Choi, Kwang-Ho (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Park, Seung-Won (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Kim, Sung-Wan (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Hwang, Jae-Sam (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Kang, Seok-Woo (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
  • 김성렬 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 구태원 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 최광호 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 박승원 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 김성완 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 황재삼 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 강석우 (농촌진흥청 국립농업과학원 농업생물부)
  • Received : 2012.04.18
  • Accepted : 2012.10.16
  • Published : 2012.12.30
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Abstract

Cecropin is a well-studied antimicrobial peptide that play important role as key factor in insect humoral immunity. In this study, cecropin-like antimicrobial peptide was isolated and purified from the larval haemolymph of immune-challenged japanese oak silkworm, Antheraea yamamai. To isolate antimicrobial peptide, we separated and compared acidic extracted hemolymph protein bends between control and immune-challenged larvae using SDS-PAGE analysis. In the immune hemolymph extract, but not of non-immune hemolymph, we detected differential expressed peptide band with molecular mass 4,223.01 Da. To understand this peptide better, we successfully purified this peptide using cation exchange chromatography and gel permeation chromatography. Its N-terminal amino acid sequence obtained by Edman degradation evidenced a significant degree of identity with other lepidopteran cecropins. The purified A. yamamai cecropin-like peptide showed a broad spectrum of activity against fungi, Gram-negative and Gram-positive bacteria.

세크로핀(cecropin)은 곤충의 체액성 면역에 있어서 효과적인 방어인자로 작용하는 항균 펩타이드로 잘 알려져 있다. 본 연구에서는 면역 유도된 천잠, Antheraea yamamai 유충 혈림프로부터 세크로핀 항균 펩타이드 분리 및 정제를 실시하였다. 먼저 항균 펩타이드를 분리하기 위해서, 면역 유도된 유충 및 정상 유충으로부터 추출된 혈림프 단백질 시료에 대한 단백질 전기영동(SDS-PAGE)를 통하여 비교분석하였다. 정상누에 혈림프 시료에 비해 면역 유도된 혈림프 추출물에서만 특이적으로 발현되는 분자량 4,223.01 Da의 펩타이드 밴드를 검출하였다. 선발된 면역유도 특이적 발현 펩타이드의 특성 분석을 위해서 이온교환 크로마토그래피 및 gel permeation 크래마토그래피을 수행하여 특이적으로 발현되는 펩타이드를 성공적으로 순수 정제하였다. 정제된 펩타이드는 Edman degradation법으로 N말단 아미노산 서열을 결정하였고 다른 나비목곤충의 세크로핀과 매우 높은 상동성을 나타내어 세크로핀으로 동정하였다. 또한 정제된 천잠 세크로핀 항균 펩타이드는 그람음성세균, 그람양성세균 및 곰팡이에 대해 폭 넓은 항균 스펙트럼을 나타냈었다.

Keywords

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