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研究生:陳虹聿
研究生(外文):Hong-yu Chen
論文名稱:短小桿菌PTB3分泌性蛋白質之構築與表達
論文名稱(外文):Construction and Expression of the Secreted Protein from Curtobacteria sp. PTB3
指導教授:洪堂耀
指導教授(外文):Tang-yao Hong
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:73
中文關鍵詞:醣基水解酵素短小桿菌分泌性蛋白
外文關鍵詞:3-glucanasebeta-1secretion proteinCurtobacterium sp.
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本論文構築的基因源自於短小桿菌(Curtobacterium sp. PTB3),此菌株是由實驗室從屏東蓮霧園土壤中,所篩選到具高beta-1,3-葡聚糖水解酵素活性的菌株,在選殖beta-1,3-葡聚糖水解酵素基因時,選殖到一段3.8 kb的DNA片段,內含有一內切型的beta-1,3-葡聚糖水解酵素基因與一段長約1.8 kb未知功能的分泌性蛋白質基因;內切型的beta-1,3-葡聚糖水解酵素基因由實驗室學姊構築表現質體並成功表達了此酵素基因,已完成酵素生化特性與抗真菌孢子萌發試驗之探討。另一段分泌性蛋白質基因在細菌中具有何種功能則尚不清楚,雖有文獻指出beta-1,3-葡聚糖水解酵素基因與真菌外切型具有同源性,但未直接以生化證據證實其酵素活性,因而本研究主要著重在此外泌性基因的構築與功能探討。此段基因長1782個鹼基,可轉譯出593個胺基酸,預測之蛋白質分子量約62.6 kDa;實驗中試驗多種載體包含了pET41a、pETDuet、pQE32、pGEX4T-1,皆接入大約1.8 kb外泌型基因,構築完成的轉形株,利用IPTG誘導蛋白質基因的表現,其中pET41a、pETDuet與pQE32之轉形株,經由SDS-PAGE分析比對,在預測位置並無發現額外的蛋白質表現,而pGEX4T-1轉形株有融合蛋白表現之,經穀胱甘肽轉移酶(GST)管柱純化後,測試所純化的融合蛋白對不同的醣類水解活性,包含澱粉、纖維素、幾丁質、葡聚醣等,但都無偵測到此融合蛋白之醣基水解酵素的活性,此分泌性蛋白質之生理活性尚待進一步探討。
The bacterial strain PTB3 of Curtobacterium sp. was isolated from wax-apple orchard in Pingtung, Taiwan. It could produce a highly enzyme activity of beta-1,3-glucanase. A 3.8 kb DNA fragment which containing beta-1,3-glucanase gene was isolated from Curtobacterium sp.. The DNA fragment was predicted contain two functional genes, an endo-type gene which could produce beta-1,3-glucanase, and a secretion gene that function was unknown. The endo-type beta-1,3-glucanase gene has been successful to expression, analysis the biochemical characterization and anti-germination activity to phytopathogenic fungal conidia. A homology search using the BLAST program found no similarity between the secreted protein and the known endo-beta-1,3-glucanases, but has significant similarity to proteins of unknown function from Streptomyces spp.. Determination of the nucleotide sequence of the DNA fragment revealed an 1782 bp ORF which may encode a protein of 592 amino acids with a calculated molecular mass of 62.6 kDa. To confirm that the ORF actually function, a DNA fragment containing the ORF was amplified by PCR and transferred into E. coli for protein expression by pET41a, pETDuet, pQE32, or pGEX4T-1 vectors. The fusion protein which was constructed on pGEX4T-1 vector was purified by glutathione S-transferase (GST) column and appeared homogeneous on SDS-PAGE. There were no detectable enzyme activity on different carbohydrate substrates including beta-glucan, starch, cellulose, chitin, and xylan by purified protein. Therefore, the physiological activity of the secreted protein remains to be discovered in further.
中文摘要 Ⅰ
Abstract Ⅱ
致謝 Ⅲ
目錄 Ⅶ
圖次 Ⅸ
表次 Ⅹ
附錄 XI
前言 1
材料與方法 8
一、實驗試藥 8
二、培養基之製備 8
三、儀器設備 9
四、菌株來源 9
五、短小桿菌分泌型基因選殖 10
1.染色體DNA的萃取 10
2.表現載體之構築 10
3. PCR-M Clean-up system kit (聚合酵素鏈鎖反應產物純化套組) 11
4. Gel-M Gel Extraction System (Gel Cutting)瓊膠萃取系統 11
5.瓊膠(agarose gel)電泳 12
6.載體DNA的製備 12
7.接合反應(Ligation) 13
8.製備勝任細胞(competent cell) 13
9.轉形(Transformation) 13
七、蛋白質之表達與純化 14
1.菌體之培養 14
2.選殖株之粗蛋白萃取 14
3.蛋白質以GST Fusion Protein System純化 14
八、純化的蛋白質生化特性分析 15
1. SDS-聚丙烯醯胺凝膠電泳(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 15
2.分泌性蛋白質活性偵測 16
結果與討論 18
一、Curtobacterium sp. PTB3基因之分析 18
二、分泌性蛋白表達質體的構築 19
1.pET41a載體構築 20
2.pETDuet載體構築 20
3.pQE32載體構築 21
三、重組之外泌性蛋白之表現 21
1.構築於pET41a表現載體的蛋白表現 21
2.構築於pETDuet表現載體的蛋白表現 22
3.構築於pQE32表現載體的蛋白表現 22
四、改變構築載體表現融合蛋白 23
五、pGEX4T-1表現融合蛋白 23
1.pGEX4T-1表現質體的構築 23
2.構築於pGEX4T-1表現載體的蛋白表現 24
3.GST融合蛋白的純化 24
六、純化之分泌性融合蛋白活性之偵測 24
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