臺灣博碩士論文加值系統

利用聚合�○s鎖反應，從樟芝子實體選殖出兩段2-Cys peroxiredoxin (AcPrx1及AcPrx) 之全長 cDNA 基因序列，全長大小分別為 942 bp 和 940 bp，而轉譯區分別為 645 bp 和 567 bp，可分別譯出 214 個及 188 個胺基酸。在以往的研究發現，2-Cys Prxs序列會包含兩個高保留的半胱胺酸 (Cys 48與170)，而 AcPrx1 (Cys 47與170) 和 AcPrx (Cys 47與165) 亦符合此特性。
以 pAVD 10 作為表現載體，轉形至 E.coli C43 (DE3) 中，再經由IPTG的誘發，即可表現出大量的重組蛋白質，而表現的重組蛋白為 AcPrx1 及 AcPrx 個別與 6 His-tagged 所形成的融合蛋白，可利用含有鎳螯合物凝膠 (nickel chelating Sepharose) 之親合性管柱來純化目標蛋白。
於10 % SDS–PAGE 可觀察到所純化的 AcPrx1 及 AcPrx 大多為雙倍體，只有極少量為單元體，若將兩者處理還原劑 DTT 時，均會使雙倍體轉變為單元體。在活性方面，顯示兩者均為 “thiol- or thioredoxin- dependent activity”，且 AcPrx1 可以保護 DNA 免於受到 .OH 的傷害。
觀察 AcPrx1 之基本特性，於 60℃下加熱 2.5 min 後，活性仍存有 60 %；處在不同 pH 的環境下發現，在 pH 5-11 的範圍內 AcPrx1 活性均很安定；而以 2 % SDS 處理，活性仍有59.5 %；若以 0.2 M imidazole處理，活性還有45 % ；加入 chymotrypsin 會迅速使AcPrx1失活，處理 20 min 後活性即剩46 %；於 trypsin 作用，則在 40 min 時活性仍有57.1 %。

Peroxiredoxins (Prxs) play important roles in antioxidation and cell signaling. A Gene encoding 2-Cys Prx isozyme (2-Cys Prx1) was identified in the expressed sequence tag data base of the Antrodia camphorata, a medicinal mushroom found only in Taiwan. Full-length cDNA of 942 bp encoding the putative 2-Cys Prx from fruiting bodies of A. cam. was cloned by PCR. The deduced amino acid sequence is well-conserved among the reported 2-Cys Prx including the two Cys residues (Cys 48 and 170). To further characterize the A. cam. 2-Cys Prx1, the coding region was subcloned into an expression vector pAVD10 and transformed into E. coli. The recombinant 6His-tagged Prx1 was expressed and purified by Ni2+-nitrilotriacetic acid Sepharose column. The purified enzyme showed two forms by 10 % SDS-PAGE. The enzyme retained 60% activity at 60℃ for 2.5 min. The enzyme was activity under a broad range of pH from 5-11. The enzyme activity lost 59.5% in the presence of 2 % SDS. The enzyme showed 57.1% activity after 40 min of incubation at 37℃ with typsin. The study of the enzyme’s properties was beneficial for the applications of Prx1 in medicine or as health food.

中文摘要……………………………………………………I
英文摘要……………………………………………………II
縮寫表………………………………………………………III
壹、前言……………………………………………………1
一、樟芝………………………………………………….1
二、自由基……………………………………………….5
三、Peroxiredoxins 的發現起源及其歷史回顧………8
四、哺乳動物的 Prx 分為三型及其個別作用機制……14
五、Prx 之調控………………………………………….16
六、2-Cys Prxs 與訊息傳遞的關係……………………19
七、研究動機…………………………………………….21
貳、實驗材料………………………………………………33
參、實驗方法………………………………………………38
一、重組表現質體 DNA 之建構…………………………38
二、重組表現質體 DNA 之轉形作用……………………41
三、重組蛋白質之表現與純化………………………….43
四、酵素活性測定……………………………………….50
五、蛋白質之基本特性分析…………………………….52
肆、結果與討論……………………………………………55
一、AcPrx1 與 AcPrx 兩段全長基因序列之比較…….55
二、AcPrx1 及 AcPrx 之分類………………………….55
三、AcPrx1 及 AcPrx 之subcloning………………….56
四、AcPrx1 及 AcPrx 之表現與純化………………….56
五、AcPrx1 及 AcPrx 活性的測定…………………….58
六、AcPrx1 特性分析……………………………………61
參考文獻……………………………………………………83
附錄…………………………………………………………91

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