파아지디스플레이 기법을 통한 TLR4/MD2의 새로운 펩타이드 라이간드 발굴
High Throughput Screening of Novel TLR4/MD2 Modulating Peptides through Phage Display System
- 주제(키워드) Innate immunity , MD2 , Phage display , Peptide , TLR4
- 발행기관 아주대학교
- 지도교수 최상돈
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000018952
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Toll-like receptors (TLRs) are one of the most important receptors for innate immunity to recognize pathogens and regulate the signaling pathway. Several mechanisms have been discovered which involved in TLRs-mediated pathways in inflammatory and autoimmune diseases, however, the therapeutics of these are still under investigation. Here, we designed phage-displayed peptide library to screen the novel TLR4/MD2 binding peptides. Phage display has been proved to be a powerful technology to screen the proteins, cells or tissues binding specific peptides. After several rounds of biopanning, high throughput screening, ELISA assay was performed and the binding affinity of selected single phage particles with TLR4/MD2 complex were measured. The phage clones showing high binding affinity with TLR4/MD2 were isolated and sequenced. The synthesized peptides antagonized LPS-induced activation of nuclear factor (NF)-κB in TLR4/MD2/CD14-transfected human embryonic kidney (HEK)-293 cells. To confirm the LPS antagonistic effect of selected peptides, the NF-κB nuclear translocation, IL-6 and NO level were investigated in peptide treated Mouse macrophage cells (RAW264.7). Extensive computational strategies were further applied to explore the possible binding pattern of TLR4/MD2 and selected peptides. These findings suggest the therapeutic potential of screened peptides against TLR4/MD2 related immune diseases but further validation in inflammatory and immune related disorders is needed.
more목차
Introduction 1
Results 4
Screening of TLR4/MD2 binding phage displayed peptides 4
Screened peptides suppress LPS-induced activation of NF-κB 4
Inhibitory effects of peptides on expressions of LPS-induced IL-6 and proinflammatory mediators 6
Protein-protein docking and MD simulation to explore the binding pattern of screened peptides in TLR4/MD2 pocket 7
Discussion 16
Materials and Methods 18
Library construction 18
Biopaning 18
High throughput screening (HTS) 19
DNA sequencing and peptide synthesis 20
Cell culture and treatment 21
NF-κB activity (SEAP) assay 21
Nuclear fractionation and Western blot analysis 22
Confocal microscopy 23
Inflammatory cytokine and NO detection 23
Homology modeling 24
Molecular dynamic (MD) simulation 24
Protein-protein interaction (Docking) 26
References 27
국문요약 35
