생명과학회지 (Journal of Life Science)

  • 제27권11호
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  • Pages.1276-1289
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  • 2017
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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참담치(Mytilus coruscus) 혈구(hemocyte)에서 분리한 McSSP-31의 항균 특성 분석

The Antimicrobial Characteristics of McSSP-31 Purified from the Hemocyte of the Hard-shelled Mussel, Mytilus coruscus

  • 오륜경 (국립수산과학원 생명공학과) ;
  • 이민정 (국립수산과학원 생명공학과) ;
  • 김영옥 (국립수산과학원 생명공학과) ;
  • 남보혜 (국립수산과학원 생명공학과) ;
  • 공희정 (국립수산과학원 생명공학과) ;
  • 김주원 (국립수산과학원 생명공학과) ;
  • 박중연 (국립수산과학원 생명공학과) ;
  • 서정길 (군산대학교 식품생명과학부) ;
  • 김동균 (국립수산과학원 생명공학과)
  • Oh, Ryunkyoung (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Lee, Min Jeong (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kim, Young-Ok (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Nam, Bo-Hye (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kong, Hee Jeong (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kim, Joo-Won (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Park, Jung-Youn (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Seo, Jung-Kil (Department of Food Science and Biotechnology, Kunsan National University) ;
  • Kim, Dong-Gyun (Biotechnology Research Division, National Institute of Fisheries Science)
  • 투고 : 2017.09.27
  • 심사 : 2017.10.19
  • 발행 : 2017.11.30
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초록

참담치 hemocyte에 존재하는 항균 펩타이드를 역상 HPLC column을 사용하여 분리 및 정제하였다. 정제된 펩타이드는 matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS) 분석을 통해 분자량이 3330.549 Da이며, edman 분해법을 통해 14개의 N-말단 아미노산 서열을 확보하였다. 분석한 N-말단 서열은 M. californianus의 sperm-specific protein Phi-1과 protamine-like PL-III protein과 각각 93%와 87%의 유사도를 나타냈으며, M. edulis의 sperm-specific protein Phi-1과 87% 일치함을 확인하였다. 또한 open-reading frame (ORF)은 306 bp의 길이에 101개의 아미노산을 코딩하고 있음을 밝혔으며, 이는 M. californianus의 sperm-specific protein Phi-1와 93.5% 유사하였다. 분자량과 아미노산 서열에 근거하여 31개 아미노산으로 구성된 펩타이드를 합성하였으며 이는 그람 양성균인 B. subtilis, S. mutans, S. aureus와 그람 음성균인 E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa 그리고 진균류인 C. albicans에 항균 활성을 보였다. 합성한 펩타이드는 항생제 내성균주인 S. aureus CCARM 0203와 S. aureus CCARM 0204에 항균 활성을 보였다. 합성 항균 펩타이드는 넙치 혈장에 대한 용혈현상은 없었고, 세포독성을 확인한 결과 HUVEC cell line에 전혀 독성을 보이지 않았다. 본 연구결과, 참담치의 혈구로부터 분리 및 정제한 sperm-specific protein 유래 항균 펩타이드는 다양한 균주에 항균 활성을 보였고 낮은 세포독성을 가졌으며, 이러한 특성은 본 실험에서 분리한 항균 펩타이드가 항생제 대체재로서 개발 가능성을 제시하고 있다.

This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.

키워드

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