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Isolation of Antagonist of Receptor Activator of NF-kappaB ligand (RANKL) from an Engineered Zinc Finger Library

Title
Isolation of Antagonist of Receptor Activator of NF-kappaB ligand (RANKL) from an Engineered Zinc Finger Library
Authors
김수연
Issue Date
2013
Department/Major
대학원 생명·약학부생명과학전공
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
심현보
Abstract
Receptor activator of NF-κB ligand (RANKL), a member of the tumor necrosis factor (TNF) family of proteins, binds to its receptor RANK on osteoclasts or osteoclast precursors and acts as an essential factor of osteoclast differentiation and activation. RANKL overproduction is implicated in the pathogenesis of osteoporosis, rheumatoid arthritis, immune system and certain cancers. Here we isolated protein clones specific for the murine RANKL from ZiFi (randomized Zif268) library. The phage display selection of ZiFi library and phage ELISA screening of panning outputs against murine RANKL gave us one clone (B7) that specifically recognized the target, and significantly inhibited osteoclast differentiation of RAW264.7 cells understimulation with murine RANKL. When RAW264.7 cells were stimulated with mRANKL, ZiFi-B7 decreased Nuclear factor of activated T cells cytoplasmic 1 (NFATc1) that is a key transcription factor of osteoclastogenesis as well as tartrate-resistant acid phosphatase (TRAP) that is known as a biochemical marker of osteoclast function and degree of bone resorption. These results suggest that a zinc finger protein with its DNA binding surface randomized is a valuable alternative scaffold that can has target specific biological activity.;종양괴사인자 단백질 중 하나인 RANKL (receptor activator of NF-κB ligand)은 파골세포 (osteoclast)나 파골세포의 전구세포에 존재하는 RANK와 결합하고, 파골세포의 분화 및 활성에 필수적인 요소로서 작용한다. RANKL의 과다 분비는 골다공증, 류머티즘 관절염, 면역체계 이상, 특정 암의 발병과 관련이 있다. 우리는 여기서 ZiFi (randomized Zif268) library로부터 murine RANKL에 특정하게 결합하는 클론들을 찾았다. ZiFi library를 이용하여, phage display selection과 이 과정을 통해 얻은 panning outputs의 phage ELISA screening을 통해 mRANKL을 특이적으로 인식하고, mRANKL의 자극으로 인한 RAW264.7 cells의 파골세포 분화를 상당히 억제하는 하나의 클론 (B7)을 얻었다. mRANKL로 RAW264.7 cells을 자극하였을 때, ZiFi-B7의 처리는 뼈 흡수 정도와 파골세포 기능의 생화학적 표지자로 알려진 TRAP (tartrate-resistant acid phosphatase)뿐만 아니라 파골세포 형성에 주요한 전사 요소인 NFATc1 (nuclear factor of activated T cells cytoplasmic 1)의 발현 또한 감소시킨다. 이러한 결과들은, 무작위화된 DNA 결합 표면을 가진 zinc finger protein이 생물학적 활성을 지닌 target-specific 클론들을 생산할 수 있는 scaffold로써 대체 가능하다는 것을 시사한다.
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