별불가사리(Patiria pectinifera) 유래 새로운 cysteine-rich Macin 의 발견과 재조합 생산을 통한 항균 활성 및 특성 연구

다국어 초록 (Multilingual Abstract)

The ocean is a reservoir of diverse life forms including various marine animals that are constantly exposed to abundant microorganisms, including pathogenic bacteria. Marine invertebrates, such as starfish, lack adaptive immunity and rely heavily on their innate immune systems to defend against these microorganisms. A key factor in the innate immune response is antimicrobial peptides (AMPs). This study is the first to identify Macin in the starfish Patiria pectinifera and to measure its antimicrobial activity. This AMP, which exhibits both antimicrobial and neuroregenerative activities, has not been previously reported in echinoderms before. This study also investigates the effects of different cysteine connectivities and immune challenges on PpMacin’s antimicrobial activity and transcriptional expression levels, respectively. The full nucleotide and amino acid (AA) sequences of PpMacin were determined through cDNA cloning, revealing a total sequence of 1527 bp, including a 5ʹ untranslated region (UTR) of 140 bp, an open reading frame (ORF) of 264 bp, and a 3ʹ UTR of 1123 bp. The ORF encodes a total of 87 AAs, including a signal peptide of 24 AAs and a mature peptide of 63 AAs. The recombinant PpMacin (rPpMacin) was successfully produced through a heterologous expression system with the pET-28a(+)-TrxA vector system and Escherichia coli BL21 (DE3). The antimicrobial activity of the three rPpMacin forms (refolded, reduced, and one missing a disulfide bond) was investigated using the ultrasensitive radial diffusion assay, with results showing that cysteine connectivity significantly affects PpMacin’s antimicrobial capacity. The bacteriolytic activity of rPpMacin was confirmed using a chromogenic plate assay. Furthermore, NPN and ONPG assays demonstrated that rPpMacin weakly interacted with bacterial membranes at the experimental concentration. The transcriptional expression levels of PpMacin in starfish tissues were evaluated using RT-qPCR, revealing high PpMacin expression in coelomocytes and coelomic epithelium. Finally, temporal expression of PpMacin following immune challenge was highest in coelomocytes at 4 h and 24 h.

목차 (Table of Contents)

• Ⅰ. 서론 1
• Ⅱ. 재료 및 방법 8
• 1. 재료 8
• 1.1 실험동물 및 조직 8
• 2.방법 8
• 2.1 Macin의 별불가사리 유래 항균성 펩타이드 후보군 선정 8
• 2.2 PpMacin의 전체 뉴클레오티드 서열 조사 9
• 2.2.1 5ʹ RACE PCR 10
• 2.2.2 3ʹ RACE PCR 12
• 2.3 In silico analysis 13
• 2.4 Recombinant production 14
• 2.4.1 Recombinant plasmid construction 14
• 2.4.2 Optimization of recombinant PpMacin expression conditions 17
• 2.4.3 Mass production, Ni-NTA purification, Dialysis 19
• 2.4.4 Cleavage of the recombinant fusion protein (TrxA-PpMacin) 21
• 2.4.5 Refolding process 22
• 2.5 In vitro antimicrobial activity assay 22
• 2.6 Analysis of the effect of cysteine linkage on antimicrobial activity 23
• 2.6.1 Reduction process 23
• 2.7 Mode of action studies 24
• 2.7.1 Chromogenic plate assay 24
• 2.7.2 Outer membrane permeability assay 25
• 2.7.3 Inner membrane permeability assay 25
• 2.8 PpMacin transcriptional expression analysis 26
• 2.8.1 Tissue distribution of PpMacin transcripts 26
• 2.8.2 Post-immune challenge 27
• Ⅲ. 결과 및 토론 31
• 1. PpMacin의 후보군 선정 및 full sequence 분석 31
• 2. Multiple alignment & 3D model of PpMacin with Macin family AMPs 35
• 3. 재조합 단백질 생산을 통한 Recombinant PpMacin 생산 39
• 4. PpMacin의 항균 활성 및 시스테인 연결에 따른 활성 변화 54
• 5. PpMacin’s mode of action 70
• 5.1 Chromogenic plate assay 70
• 5.2 N-Phenyl-1-naphthylamine (NPN) uptake assay 71
• 5.3 ortho-Nitrophenyl-β-D-galactopyranoside (ONPG) assay 71
• 6. PpMacin의 발현량 비교 77
• 6.1 별불가사리 조직별 PpMacin 전사체 발현량 비교 77
• 6.2 Immune challenge 후 조직별 PpMacin 발현량 비교 78
• Ⅳ. 참고 문헌 84
• 논문 요약 90
• Acknowledgement 92