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研究生:陳韻帆
研究生(外文):Yun-Fan Chen
論文名稱:Bacillussp.P-6中性金屬蛋白基因的選殖、定序及其基因特性之探討
論文名稱(外文):Cloning, Sequencing and Characterization of Neutral Metalloprotease Gene from Bacillus sp. P-6
指導教授:蔣啟玲蔣啟玲引用關係
指導教授(外文):Chii-Ling Jeang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:100
中文關鍵詞:基因的選殖定序基因特性中性金屬蛋白枯草桿菌屬
外文關鍵詞:CloningSequencingCharacterizationNeutral Metalloprotease GeneBacillus sp.pro-sequnece function
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中文摘要
本研究藉由shotgun-cloning方式,以E. coli DH5α為宿主細胞pBluescript II KS(-)為載體構築一Bacillus sp. P-6基因庫,並由一萬多株轉型株中篩選得到一具有蛋白活性之選殖株,此選殖株質體命名為pBBNP-7。由DNA序列分析結果顯示,此選殖基因片段含有一1,698 bp 的open reading frame (ORF) 可轉譯出566個胺基酸,推算其分子量約為63 kDa。此轉譯出的蛋白質經胺基酸序列比對結果預測可分為三部份,分別為N端的pre-sequence(signal peptide)、pro-sequence 和mature protease,其中預測的mature protease 與野生株所純化出的protease分子量相同,西方墨漬法結果亦相符。將選殖出的DNA片段,依所包含之上述三個多部份,分別構築成重組表現載體pET2PM, pETProM, pETM,於E. coli中以低溫誘導蛋白質,實驗中只有E. coli BL21(DE3)/pET2PM之上清液具有蛋白活性(40 U/ml),而E. coli BL21(DE3)/pETProM、E. coli BL21(DE3)/pETM之上清液則不具蛋白活性,此結果顯示pre-sequence與pro-sequence兩者對於活體內(in vivo)蛋白之熟成(processing)缺一不可,且蛋白之熟成作用應在蛋白質送至胞壁空間後才進行。另構築雙向載體pHBNP,將完整蛋白基因包含原有的啟動子送入Bacillus subtilis DB430進行表現,於LB培養基中培養24小時後,上清液中之蛋白活性約95 U/ml;在營養限制之SSY培養基中培養36小時則可得115 U/ml之蛋白活性。

Abstract
Employing pBluescript II KS(-) as a cloning vector, the genomic library of Bacillus sp. P-6 was constructed in E. coli DH5a by way of shotgun-cloning approach. A candidate possessing protease activity was screened among approximately ten thousands of colonies by skim milk contained plate. The recombinant plasmid of the candidate was named as pBBNP-7. Analyzing the DNA sequence of the inserted DNA fragment, a 1,698 bp open read frame encoded a 566 amino acid of protein was predicted and its putative molecular mass was 63 kDa. According to the amino acid sequence alignment, the primary structure of deduced protein could be classified into three portions, since from N terminus, pre-sequence, pro-sequence, and mature protease. The deduced molecular mass of mature protease was consistent with the protease purified from Bacillus sp. P-6. and also validated by Western blotting. In the protein expression experiment at low temperature, protease activity was detected (40 U/ml) in the culture broth in E. coli BL21(DE3)/pET2PM, however, there were no protease activity detected in E. coli BL21(DE3)/pETProM and E. coli BL21(DE3)/pETM. These results suggested that pre-sequence and pro-sequence were simultaneously required for protein maturation in vivo, and the maturation was occurred after protein secreted into periplasmic space. A protease expression vector pHBNP, bearing native promoter and capable of shuttling E. coli and Bacillus subtilis, was also constructed in this study. Cultivated the Bacillus subtilis DB430/ pHBNP in LB medium, there was a maximal 95 U/ml of protease activity detected. While, a maximal 115 U/ml of protease activity detected, cultivated in nutrient limited medium SSY.

目 錄 頁數中文摘要 ------------------------------------------------------------------ i英文摘要 ------------------------------------------------------------------ ii壹、 前言 -------------------------------------------------------------- 1一、 蛋白的簡介 -------------------------------------------------- 1二、 蛋白的分類依據與其命名 -------------------------------- 1三、 微生物來源之蛋白(Microbial protease) -------------- 2四、 細菌的蛋白質分泌系統簡介 -------------------------------- 6五、 微生物分泌性蛋白的特性 -------------------------------- 8六、 Pro-sequence在蛋白中所扮演的角色 ------------------ 11七、 本實驗研究緣起與目的 -------------------------------------- 17貳、 材料與方法 ----------------------------------------------------- 18一、 菌種及質體 ----------------------------------------------------- 19二、 藥品與器材 ----------------------------------------------------- 21三、 自大腸桿菌抽取質體 ----------------------------------------- 22四、 自枯草桿菌抽取質體 ----------------------------------------- 22五、 DNA分子之電泳、剪切、回收、補齊及黏合 ---------- 24六、 大腸桿菌之轉型 — 電轉形法 ------------------------------- 24七、 枯草桿菌之轉型 — 電轉形法 ------------------------------- 24八、 Bacillus sp. P-6 染色體DNA質體基因庫之建立與NPr基因之選殖 --------------------------------------------------------------- 251. Bacillus sp.P-6 染色體DNA之製備 ----------------------- 252. 以Sau3AI將染色體DNA作不完全剪切 ----------------- 253. 質體基因庫之建立 --------------------------------------------- 264. NPr 蛋白基因之篩選與印證 ------------------------------ 26九、 NPr蛋白基因之次選殖 ------------------------------------- 27十、 NPr蛋白基因之DNA定序、電腦整理與分析 -------- 27十一、 NPr蛋白基因大量表現載體之構築 ---------------------- 271. 表現載體pET2PM之構築 ------------------------------------ 282. 表現載體pETProM --------------------------------------------- 283. 表現載體pETM ------------------------------------------------- 284. 表現載體pETPro ------------------------------------------------ 29十二、 NPr基因在E. coli 中之表現 --------------------------------- 291. 蛋白質之誘生 -------------------------------------------------- 29(1). IPTG誘導的劑量反應 .------------------------------------------ 29(2). 不同溫度誘生蛋白質 ------------------------------------------ 292. 西方轉漬法 ------------------------------------------------------ 303. 蛋白活性之分析 --------------------------------------------- 314. 蛋白質濃度測定 ------------------------------------------------ 31十三、 融合蛋白之純化 ------------------------------------------------ 311. 變性狀態:不溶性蛋白質之純化 --------------------------- 312. 原態:可溶性蛋白質之純化 --------------------------------- 32十四、 NPr蛋白基因抗體之製備 --------------------------------- 331. 抗原製備 --------------------------------------------------------- 332. 雞蛋卵黃免疫球蛋白IgY之純化 --------------------------- 34十五、 Bacillus sp. P-6 neutral metal protease 在Bacillus subtilis DB430 之表現 -------------------------------------------------- 341. 表現載體pHBNP 之構築 ------------------------------------- 342. NPr 基因表現於B. subtilis 之活性分析 ------------------- 34參、 結果 ----------------------------------------------------------------- 35一、 Bacillus sp.P-6 neutral metal protease 基因選殖 --------- 351. Chromosome 以Sau3AI 作partial digestion ------------- 372. 質體pBBNP限制圖譜之建立 ----------------------------- 383. 質體pBBNP 中蛋白基因的次選殖 ---------------------- 394. 蛋白基因之DNA定序 -------------------------------------- 415. 蛋白基因之序列分析與比對結果 -------------------------- 47二、 Bacillus sp. P-6 neutral mental protease 抗體之純化 ------- 471. 蛋白基因在E. coli大量表現載體構築 ------------------ 472. 蛋白基因大量表現及純化 --------------------------------- 533. 雞蛋卵黃免疫球蛋白之純化與西方墨漬免疫反應分析----- 59三、 Bacillus sp. P-6 neutral metal protease在E. coli中基因特性之探討--------------------------------------------------------------------- 591. E. coli 大量表現載體之構築 --------------------------------- 602. 蛋白質在E. coli 中大量表現之結果 ----------------------- 603. 不同培養條件對蛋白NPr表現的影響 ------------------ 63(1) 不同IPTG 濃度誘生蛋白質 --------------------------------- 63(2) 不同溫度下誘生蛋白質 --------------------------------------- 634. 訊息(Pre-sequence)在蛋白熟成作用(processing)所扮演之角色 ------------------------------------------------------- 65(1) 活性偵測 --------------------------------------------------------- 68(2) 蛋白表現位置之偵測 ------------------------------------------ 71四、 Bacillus sp.P-6 neutral metal protease 在Bacillus subtilis DB430 之表現 -------------------------------------------------- 731. npr基因表現載體pHBNP 之構築 --------------------------- 732. npr基因表現於B. subtilis 之活性分析 ---------------------- 75肆、 討論 --------------------------------------------------------------- 771. Bacillus sp. P- 6染色體DNA基因庫之建立與蛋白基因選殖2. Bacillus sp. P-6 neutral metalloprotease在E. coli中基因特性之探討 -------------------------------------------------------------- 773. Bacillus sp. P-6 neutral metalloprotease(Npr)在Bacillus subtilis DB430 之表現 -------------------------------------------------- 79伍、 結論 ---------------------------------------------------------------- 81陸、 參考文獻 --------------------------------------------------------- 83附錄一 --------------------------------------------------------------------- 98附錄二 -------------------------------------------------------------------- 100

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