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標題: Construction of phage-display peptide library for the screening of antibody epitope
作者: 曾斯偉
Tseng, Sy-Woei
關鍵字: phage display system;噬菌體展示技術;random peptide library;autoimmunity disease;SLE;自體免疫疾病;紅斑性狼蒼
出版社: 分子生物學研究所
Phage display technology, consisting of the expression of target sequence in the surface of phage particles and the affinity selection, is a biological system that facilitates the cloning and rapid selection of peptides or proteins from large combinatorial libraries. Libraries are generated by cloning of a batch of DNA encoding millions of variants of certain ligands into the phage genome or phagemid as a fusion to the gene encoding one of the phage coat proteins. Upon expression, the fusion coat protein is incorporated into new phage particles that are assembled in the bacterium and consequently presented on the phage surface, with its genetic material residing within the phage particles. The purpose of this study is to construct a random peptide library containing both linear and conformational epitopes to serve as an “all purpose” peptide source for searching of disease-related peptides. To achieve this goal, EcoRI-HindIII DNA fragments encoding 45 amino acid residues, of which 36 have random sequences, were cloned into the corresponding sites of the modified pCANTAB5E plasmid and the resulted plasmids were transformed into E. coli TG1. The percentage of insert-containing colonies in the transformants was evaluated by PCR using primers flanking the insert DNAs. Approximately, 83% of the clones contained inserts. The expression of the insert gene was determined by colony immunoblot analysis of a portion of the original clones using the antibody specific for E-Tag which is located downstream of the insert. The number of clone capable of expression coincided with the percentage of the insert-containing clones. Finally, the diversity of the library was evaluated by sequencing of 10 randomly picked clones. The results indicated that all exhibited difference sequences. The diversity of the library was estimated to be 4.4 x 106 according to the above information,. To establish a system for identification of disease-related peptides, affinity selection was performed using sera from system lupus erythematosus patients.

噬菌體展示技術是一種基因表現產物與親和選擇(affinity selection)結合的技術。它是將外源DNA與噬菌體外套蛋白基因透過接頭(linker)相連,進而使外源DNA主導之產物與外套蛋白融合,並展示在噬菌體表面;當以一組隨機編碼序列或基因群插入噬菌體載體進行表現及展示時,其總體即稱為噬菌體展示庫(phage display library)。所需要的目標產物,可經由與選擇體(selector)的結合而被吸附下來;被吸附的重組噬菌體可經由再感染而擴增;展示月生 月太 或蛋白的氨基酸序列則可以核酸定序重組噬菌體之基因組解讀出來。本論文的目標是構築一個含括直線與構型抗原表位月生 月太 庫,建立一個搜尋疾病相關抗原表位(epitope)之系統。由於長的月生 月太 較可能形成獨立構形、有較多的接觸位,每一分子上又有許多線性抗原表位,因此長的隨機月生 月太 庫應該是一個較好的抗原表位來源。首先將pCANTAB5E載體上的SfiI與NotI切位分別修改為EcoRI與HindIII,再將主導45個胺基酸的EcoRI-HindIII DNA片段插入修改後的pCANTAB5E載體,送入E. coli TG1菌體。以PCR檢測發現所構築的library裡有83%(33/40)帶有插上之DNA片段。再以anti-E-tag抗體進行colony immunoblot分析,結果顯示有E-tag表現的菌落數與帶有插入DNA之殖系百分比相當。由於E-tag位於插入DNA之下游,因此E-tag之表現證明插入的外源
月生 月太 的存在。最後隨機挑取10株帶有插入DNA的質體菌落,抽取其質體DNA進行核酸定序,結果都具有不同的序列。經以上方法確定所建構的月生 月太 庫至少包含4.4 x106不同的月生 月太 種類。續以紅斑性狼瘡(SLE)病患之血清對此月生 月太 庫進行親和選擇,以建立以病人血清搜尋疾病相關月生 月太 標誌的系統。
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