별불가사리(Patiria pectinifera)의 아가미와 껍질로부터 항균성 펩타이드의 정제, 특성 연구 및 재조합 펩타이드 생산 = Purification, Characterization, and Recombinant Peptide Production of Antimicrobial Peptides from the Shells containing Gills of Starfish, Patiria pectinifera

다국어 초록 (Multilingual Abstract)

Marine animals are equipped with a potent defense system, possibly due to the microbe-rich environment of their surroundings. These animals, particularly marine invertebrates like the starfish Patiria pectinifera, lack adaptive immunity and primarily rely on innate immunity. Nevertheless, they successfully protect themselves against pathogenic infections by effectively utilizing antimicrobial peptides (AMPs). These peptides play an essential role in the defense system of marine invertebrates and have gained significant attention as potential antimicrobial agents. In this study, we isolated and purified two antimicrobial peptides from the shell tissue, including the gills of Patiria pectinifera. Their antimicrobial activity was subsequently confirmed. The purified peptides were named as PpSAMP1 and PpSAMP2, with molecular weights of 4016.5177 [M+H]+ and, 4121.6002 [M+H]+ respectively. Among of them, PpSAMP1 consists of 39 amino acid residues, and the complete 877bp nucleotide sequence was confirmed through cDNA cloning. Although no significant homology with previously identified AMPs from marine invertebrates was observed, we confirmed that PpSAMP1 is similar to PpCrAMP, which was previously studied in our laboratory. It was confirmed that they belong to the same cysteine-rich AMPs family. The expression analysis of PpSAMP1 precursor transcripts confirmed that the expression level is notably high in the tube feet and coelomic epithelium. Furthermore, through ORF amplification, various isoforms of PpSAMP2 were identified, all of which were found to have a structure similar to that of PpSAMP1. The discovery of PpSAMP1 and PpCrAMP, as well as various AMPs in different echinoderm species, is expected to provide a foundation for the development of new antimicrobial drugs.

목차 (Table of Contents)

• Ⅰ. 서론 1
• Ⅱ. 재료 및 방법 7
• 1. 재료 7
• 1.1 실험동물 및 조직 7
• 1.2 시약 및 재료 7
• 2. 방법 8
• 2.1 별불가사리 아가미와 껍질 조직으로부터 추출물 조제 8
• 2.2 항균 활성 측정 9
• 2.2.1 사용 균주 및 배양 조건 9
• 2.2.2 Ultrasensitive radial diffusion assay (URDA) 10
• 2.3 항균 활성 물질의 정제 11
• 2.3.1 고상 추출을 이용한 물질의 분리 11
• 2.3.2 High performance liquid chromatography (HPLC)를 사용한 항균 활성 물질의 정제 12
• 2.3.2.1 Fraction 1로부터 PpSAMP1의 정제 과정 13
• 2.3.2.2 Fraction 2로부터 PpSAMP2의 정제 과정 14
• 2.4 PpSAMP1과 PpSAMP2의 분자량 측정 15
• 2.5 PpSAMP1과 PpSAMP2의 1차 구조 및 Homology 분석 15
• 2.6 PpSAMP1과 PpSAMP2의 cDNA cloning 15
• 2.6.1 PpSAMP1의 3′ RACE PCR 17
• 2.6.2 PpSAMP1의 5′ RACE PCR 18
• 2.6.3 PpSAMP1의 ORF amplification 18
• 2.6.4 PpSAMP2의 ORF amplification 19
• 2.6.5 RACE PCR product의 cloning 20
• 2.6.6 ORF amplification product의 cloning 21
• 2.7 In silico 분석 22
• 2.8 재조합 펩타이드 생산 23
• 2.8.1 Construction of PpSAMP1 Recombinant plasmid 23
• 2.8.2 Optimal condition for recombinant PpSAMP1 overexpression 25
• 2.8.3 대용량 생산 및 투석 27
• 2.8.4 CNBr 처리를 통한 rPpSAMP1 분리 28
• 2.9 RT-qPCR을 통한 PpSAMP1의 발현량 비교 29
• 2.10 통계처리 30
• Ⅲ. 결과 35
• 1. 별불가사리 아가미와 껍질 추출물의 항균 활성 35
• 2. 항균 활성 펩타이드의 정제 41
• 2.1 Fraction1로부터 PpSAMP1의 정제 41
• 2.2 Fraction2로부터 PpSAMP2의 정제 42
• 3. 정제된 펩타이드의 특성 분석 52
• 3.1 PpSAMP1 52
• 3.1.1 분자량 및 아미노산 서열 분석 52
• 3.2 PpSAMP2 52
• 3.2.1 분자량 및 아미노산 서열 분석 52
• 4. PpSAMP1의 전체 염기서열 분석 53
• 5. Homology 분석 54
• 6. 재조합 펩타이드 발현 60
• 7. 재조합 펩타이드 HPLC 정제 및 분자량 측정 60
• 8. Native와 재조합 펩타이드 rPpSAMP1의 retention time 비교 61
• 9. rPpSAMP1의 항균 활성 61
• 10. 조직별 PpSAMP1의 발현량 비교 63
• 11. PpSAMP2의 ORF amplification을 통한 isotype 확인 64
• Ⅳ. 고찰 72
• Ⅴ. 참고 문헌 91
• 논문 요약 100
• Acknowledgement 102