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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.author | Yin-Chen Tzeng | en |
dc.contributor.author | 曾尹貞 | zh_TW |
dc.date.accessioned | 2021-07-01T08:19:57Z | - |
dc.date.available | 2021-07-01T08:19:57Z | - |
dc.date.issued | 1997 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76292 | - |
dc.description.abstract | 利用PCR的方式,將選殖之水稻含錳超氧歧化?(MnSOD)基因的cDNA序列大量增殖,並接進表現載體pGEX-4T-1中,再送入大腸桿菌DH5α的菌株裡表現。建構好的質體經由Enzyme digestion、Southern、PCR及DNA序列分析後確定接入且無誤。細菌培養後,以0.1mM IPTG誘導GST-MnSOD融合蛋白質大量表現,並經由親和層析純化,得到均質的GST-MnSOD。但GST-MnSOD大部份會形成inclusion body,其可以6M尿素處理及透析後,回收部份GST-MnSOD。 GST-MnSOD經過thrombin處理後,可得到比水稻的含錳超氧歧化?多五個胺基酸的重組型MnSOD(recombinant MnSOD, rMnSOD)。GST-MnSOD及rMnSOD兩種蛋白質,均具有酵素活性,抑制劑H2O2及KCN不會抑制其活性,故兩者仍保有MnSOD的活性。以10% SDS聚丙烯醯胺膠體電泳分析其單元體分子量約各為50kDa、23kDa;以5-20%梯度聚丙烯醯胺膠體電泳的結果,推測兩種蛋白質都以二元體的形式存在。以等電焦集電泳分析GST-MnSOD的pI值介於pH 4.74-4.97之間,而rMnSOD則約為pH 4.64。兩種蛋白質均可耐熱60℃、20分鐘,但於80℃則幾乎不具活性;在鹼性環境中較穩定,而於pH 4.0以下則明顯失去活性。 在探討水稻MnSOD對於T7 RNA polymerase離體轉錄效率的影響時,發現GST蛋白質能提昇RNA產量達24倍之多,SOD卻不會影響轉錄效率。 | zh_TW |
dc.description.abstract | Mn-superoxide dismutase (MnSOD) had been cloned from rice cDNA library. PCR was used to insert the DNA (mmnsod) coding for the rice mature MnSOD protein into the pGEX-4T-1 expression vector. The recombinant DNA was transformed to Escherichia coli DH5α and the construct was identified by enzyme digestion, southern, PCR and DNA sequence analysis. Expression of GST-MnSOD fusion protein was induced by adding 0.1 mM IPTG to bacterial cultures and the homo-geneous GST-MnSOD was purified by the GST-glutathione affinity system. But most of GST-MnSOD protein in E. coli resulted in an insoluble aggregate which could be recovered by 6M urea and dialysis. Thrombin treatment could cleave GST-MnSOD into GST and rMnSOD (recombinant MnSOD, which had extra five amino acids in N terminal sequence than rice instric MnSOD). Both purified GST-MnSOD and rMnSOD had SOD activity, and were still insensitive to KCN and H2O2, which were characteristics of MnSOD. 10% SDS polyacrylamide gel showed that the monomer molecular weight of the two proteins was 50 kDa and 23 kDa. The native form, estimated by 5-20% gradient polyacrylamide gel electrophoresis, was dimer both in GST-MnSOD and rMnSOD. The isoelectric point of rMnSOD was 4.64, but GST-MnSOD was in the range of pH 4.74 - pH 4.97. The SOD activity declined to 30 % when the two protein incubated at 60℃ for 20 minutes, but more stable at alkaline pH environment. In vitro transcription containing T7 RNA polymerase and [α-35S] CTP are used to estimate the RNA relative products. The results indicated that GST (glutathione-S-transferase) can dramatically enhance the transcription, but SOD showed no this function. | en |
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dc.description.tableofcontents | 目 錄………………………………………………………………………Ⅰ 縮寫表……………………………………………………………………Ⅲ 中文摘要…………………………………………………………………Ⅴ 英文摘要…………………………………………………………………Ⅵ 第一章 前言………………………………………………………………1 第一節 氧自由基…………………………………………………………1 第二節 生物體的氧化逆境防禦機制……………………………………2 第三節 含錳超氧歧化?的研究…………………………………………4 第四節 THR衰減子與T7 RNA POLYMERASE………………………………6 第五節 本論文研究的方向………………………………………………7 第二章 材料與方法………………………………………………………11 第一節 將水稻含錳超氧歧化?導入大腸桿菌中………………………11 1.1 插入物的合成及前處理……………………………………………11 1.2 載體的處理…………………………………………………………13 1.3 粘接反應……………………………………………………………13 1.4 轉型…………………………………………………………………14 1.5 小量抽取質體DNA……………………………………………………16 1.6 南方氏轉印法………………………………………………………18 第二節 表現及純化蛋白質………………………………………………22 2.1 誘導蛋白質表現……………………………………………………22 2.2 純化GST融合蛋白質…………………………………………………23 2.3 尿素處理……………………………………………………………25 2.4 透析法………………………………………………………………26 2.5 總蛋白質定量法……………………………………………………26 2.6 聚丙烯醯胺電泳分析法(PAGE)…………………………………28 2.7 超氧歧化?膠片上活性染色及定量法……………………………29 2.8 蛋白質CBR染色法……………………………………………………31 第三節 水稻含錳超氧歧化?的生化定性分析…………………………31 3.1 凝血酵素的處理…………………………………………………31 3.2 抑制劑的處理……………………………………………………32 3.3 等電焦集電泳……………………………………………………32 3.4 熱處理……………………………………………………………34 3.5 pH處理……………………………………………………………35 第四節 GST-MNSOD對離體轉錄的影響……………………………………36 4.1 範本的前處理……………………………………………………36 4.2 離體轉錄…………………………………………………………37 4.3 RNA電泳分析法……………………………………………………38 4.4 終止效率(termination efficiency)計算方式……………40 第三章 結果………………………………………………………………42 第一節 將水稻含錳超氧歧化?導入大腸桿菌中………………………42 (一)以PCR合成插入物…………………………………………………42 (二)PCR產物及載體的前處理…………………………………………45 (三)粘接反應與轉型……………………………………………………45 (四)限制?處理:………………………………………………………45 (五)南方轉印法…………………………………………………………49 (六)DNA序列分析………………………………………………………50 第二節 表現及純化蛋白質………………………………………………50 (一)誘導蛋白質表現……………………………………………………50 (二)純化GST-MnSOD蛋白質……………………………………………52 (三)從inclusion body中回收GST-MnSOD……………………………52 (四)純化表………………………………………………………………56 第三節 水稻含錳超氧歧化?的生化性質分析…………………………60 (一)凝血酵素(thrombin)的處理……………………………………60 (二)原態分子量的測定…………………………………………………60 (三)對抑制劑耐受性的測試……………………………………………63 (四)pI值的測定…………………………………………………………63 (五)耐熱性測試…………………………………………………………63 (六)酸鹼度耐受性的測試………………………………………………63 第四節 GST-MNSOD對離體轉錄的影響……………………………………68 第四章 討論…………………………………………………………………82 第一節 水稻MNSOD構築至表現載體中……………………………………82 第二節 表現及純化…………………………………………………………83 第三節 生化性質分析………………………………………………………84 第四節 GST-MNSOD對離體轉錄的影響……………………………………86 第五節 未來的展望…………………………………………………………88 第五章 參考文獻……………………………………………………………89 | |
dc.language.iso | zh-TW | |
dc.title | 水稻含錳超氧歧化?在細菌中之表達及性質研究 | zh_TW |
dc.title | Overexpression and Characterization of Rice Manganese Superoxide Dismutase in E. coli | en |
dc.date.schoolyear | 85-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 109 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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