TKU015係一株以蝦殼粉為主要碳/氮源，篩選自台灣北部土壤，經鑑定為Pseudomonas 屬新種之幾丁質酶、幾丁聚醣酶和納豆激酶生產菌。TKU015生產納豆激酶之較適條件為含1 % 蝦殼粉、0.1 % K2HPO4及0.05 % MgSO4．7H2O (pH7)於30℃搖瓶培養2天。SDS-PAGE測得分子量為21 kDa。納豆激酶活性受PMSF完全抑制，Fe 2+ 則會提高其活性。於基質特異性方面，因對N-succinyl-Ala-Ala-Pro-Phe-pNA的感受性較高，而將之歸納為類似凝乳蛋白的絲胺酸型蛋白酶。
由液相層析串聯式質譜分析得到納豆激酶的胺基酸序列片段，經比對有23 %序列接近Bacillus cereus ATCC 14597的幾丁質結合蛋白。設計適當的引子，經由一連串的聚合酶鏈鎖反應，發現TKU015的殺蟲蛋白基因序列片段。經由胺基酸序列比對，與Pseudomonas entomophila L48序列相近，設計合宜的引子以聚合酶鏈鎖反應，找到殺蟲蛋白基因的C端。並且藉由GenomeWalkerTM和聚合酶鏈鎖反應找出殺蟲蛋白的基因全長2079核苷酸單位，並進一步做基因選殖。首先將基因轉殖到pET-32 Xa/LIC的載體上，轉形入大腸桿菌XL1-Blue宿主中，利用XL1-Blue宿主特性將質體完整建構，之後萃取純化質體再轉形入大腸桿菌BL21宿主中表現，用IPTG誘導表現於37℃搖瓶培養1天，可表現94.7 kDa的蛋白質產物。用HisTag將蛋白質純化，去純多餘的鹽類，之後利用factor Xa protease 將目標蛋白質77 kDa進一步分離出來，得到的蛋白質對果蠅幼蟲做生物活性測試。

The chitinase, chitosanase and nattokinase producing strain, Pseudomonas sp. TKU015, was isolated from the soil in North Taiwan. The shrimp shell waste powder was used as sole carbon / nitrogen source. The optimized condition for nattokinase production was found when the culture was shaken at 30℃for 2 days in 100mL of medium contain 1% SSP, 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH7). The molecular mass of the nattokinase determined by SDS-PAGE was approximately 21 kDa. The nattokinase was inhibited completely by PMSF, and more activated by Fe2+. The most sensitive substrate for nattokinase was N-succinyl-Ala-Ala-Pro-Phe-pNA. Therefore, nattokinase was considered to be a chymotrypsin-like serine protease.

The results of peptide mass mapping showed that two tryptic peptides of the nattokinase were identical to a chitin binding protein from Bacillus cereus ATCC 14579 (GenBank accession number gi30020946) with 23% sequence coverage. Designing the suitable primer, by way of a succession of polymerase chain reaction, discovered the insecticidal protein gene sequence fragment. The sequence was similar that amino acid sequence comparison with Pseudomonas entomophila L48, then designing that suitable that, by way of a succession of polymerase chain reaction, found the C end of insecticidal protein gene. The all insecticidal protein gene of 2079 base pair was revealed that used GenomeWalkerTM and polymerase chain reaction.
Cloning insecticidal protein gene from Pseudomonas sp. TKU015 in the pET-32 Xa/Lic vector, and transferred to the Escherichia coli XL1-Blue host. The plasmid constructed completely because of the host characteristic. Afterward purification plasmid was extracted and then expression of transferred the Escherichia coli BL21 host. Inducing the performance of 94.7kDa the protein product with IPTG when the culture was shaken at 37℃for 1 day. The purification of insecticidal protein used HisTaq, and then desalted. Finally, the 77 kDa of goal protein was separated by using factor Xa protease. The protein was to be the biological activity test against larva of fruit fly.

目錄
	頁次

簽名頁
授權書
中文摘要	I
英文摘要 III
誌謝	V
目錄	VI
圖目錄	XI
表目錄	XIII

第一章 緒論	1
第二章 文獻回顧	2
     2.1 菌種之簡介	2
     2.2 幾丁質之應用	2
     2.3 納豆激酶	3
        2.3.1 納豆激酶溶解血栓之功用	3
     2.4 化學性與生物性農藥	4
2.4.1 化學農藥	4
2.4.2 生物農藥	4
     2.5 生物防治之微生物應用	6
2.5.1 細菌性殺蟲劑	6
  2.5.1.1 蘇力菌	6
  2.5.1.2 其他細菌防治昆蟲的特性	7
2.5.2 Pseudomonas spp.殺蟲物質	8
2.5.3 Pseudomonas spp.殺蟲蛋白之比較	9
2.6 利用生物技術轉殖殺蟲基因	9
第三章 材料與方法	13
     3.1 實驗菌株	13
     3.2 選殖載體	13
     3.3 實驗材料	13
     3.4 實驗儀器	15
     3.5 酵素最適培養條件探討	15
3.5.1 碳/氮源之選擇	15
3.5.2 碳/氮源濃度之影響	16
3.5.3 培養溫度	16
3.5.4 培養體積 (通氣量)	16
3.5.5 培養時間	16
     3.6 酵素之分離純化	16
3.6.1 粗酵素液之製備	17
3.6.2 陰離子交換層析	17
     3.7 納豆激酶之活性測定	17
     3.8 蛋白質電泳分析	18
     3.9 蛋白質定量分析	18
     3.10 酵素之特性分析	19
        3.10.1 金屬離子與抑制劑對酵素活性之影響	19
        3.10.2 納豆激酶之基質特異性	19
        3.10.3 液相層析串聯式質譜分析納豆激酶	19
     3.11 聚合酶鏈鎖反應鑑定殺蟲蛋白基因	20
3.11.1 設計引子	20
3.11.2 聚合酶鏈鎖反應	20
     3.12 殺蟲蛋白基因全長	21
3.12.1 應用GenomeWalkerTM建立基因庫	21
3.12.2 基因特異性引子合成	21
3.12.3 聚合酶鏈鎖反應	21
     3.13 殺蟲蛋白基因選殖	22
        3.13.1 設計引子	22
        3.13.2 聚合酶鏈鎖反應	23
        3.13.3 接合到pET32 Xa/LIC載體上	23
        3.13.4 轉形	23
     3.14 殺蟲蛋白	24
        3.14.1 誘導產生蛋白質	24
        3.14.2 不溶性內涵體蛋白	25
        3.14.3 純化殺蟲蛋白	25
     3.15 殺蟲蛋白生物活性測試	26
        3.15.1 供試昆蟲	26
        3.15.2 生物活性測試	26
第四章 結果與討論	27
     4.1 納豆激酶生產菌之篩選	27
     4.2 菌株之特性	27
     4.3 碳/氮源之選擇	27
     4.4 納豆激酶較適生長條件探討	27
     4.5 納豆激酶之分離純化	28
        4.5.1 粗酵素液製備	28
        4.5.2 陰離子交換層析	28
        4.5.3 綜合結果	29
     4.6 納豆激酶之分子量測定	29
        4.6.1 SDS-PAGE	29
        4.6.2 綜合結果	29
     4.7 納豆激酶之特性分析	30
        4.7.1 金屬離子對納豆激酶之影響	30
        4.7.2 納豆激酶之基質特異性	30
        4.7.3 納豆激酶的液相層析串聯式質譜分析	31
     4.8 Pseudomonas sp.TKU015殺蟲蛋白	44
        4.8.1 殺蟲蛋白基因序列	44
        4.8.2 應用GenomeWalkerTM找出殺蟲蛋白基因序列全長	44
     4.9 Pseudomonas sp.TKU015殺蟲蛋白基因選殖	45
        4.9.1 殺蟲蛋白基因接合在pET32 Xa/LIC載體上	45
        4.9.2 基因轉形	45
     4.10 生物活性測試	46
第五章 結論	61
參考文獻	63
附件	73

圖目錄
	                                           頁次

圖2.1 納豆激酶在血栓溶解系統中之直接與間接作用	11
圖4.1 Pseudomonas sp.TKU015之顯微鏡照片	32
圖4.2 16S rDNA核酸序列分析	33
圖4.3 SSP濃度對TKU015納豆激酶生產之影響	34
圖4.4 培養溫度對TKU015納豆激酶活性生產之影響	35
圖4.5 納豆激酶之DEAE-Sepharose CL-6B層析圖譜	36
圖4.6 各純化步驟之SDS-PAGE電泳分析圖	38
圖4.7 由胺基酸序列片段地圖設計p203 及p206 引子，對 Pseudomonas sp.TKU015 進行聚合酶連鎖反應所得產物之電泳圖	47
圖4.8 以p239 及p240 引子，對Pseudomonas sp.TKU015 進行聚合酶連鎖反應所得產物之電泳圖	48
圖4.9 GenomeWalker™ 應用四種限制酶作用於Pseudomonas sp.TKU015之實驗流程圖	49
圖4.10 GenomeWalker 資料庫和基因特異性引子GSP1 (p267)、GSP2 (p268)試圖找出殺蟲蛋白基因全長	50
圖4.11 GenomeWalker 資料庫和基因特異性引子GSP1(p274)、GSP2(p276)找出殺蟲蛋白基因全長	51
圖4.12 Pseudomonas sp.TKU015殺蟲蛋白胺基酸序列全長	52
圖4.13 Pseudomonas sp.TKU015殺蟲蛋白與Pseudomonas entomophila L48 殺蟲蛋白序列比對	53
圖4.14 設計p282 及p283 引子進行聚合酶連鎖反應，從Pseudomonas sp.TKU015 genomic DNA 找到殺蟲蛋白基因全長並且萃取DNA.
        	54
圖4.15 設計p291及p292引子擴增TKU015殺蟲蛋白。並選殖於pET32
        Xa/LIC載體	55
圖4.16 Pseudomonas sp.TKU015殺蟲蛋白基因選殖實驗流程圖 56
圖4.17各純化步驟之SDS-PAGE電泳分析圖	57
圖4.18 Xa蛋白酶處理殺蟲蛋白之SDS-PAGE電泳分析圖	58
圖4.19 TKU015殺蟲蛋白對於果蠅幼蟲之致死率	59
圖4.20 殺蟲蛋白質之基因圖譜	60

表目錄
	                          頁次

表2.1 拮抗微生物之種類與其防治機制	12
表4.1 TKU015納豆激酶之純化	37
表4.2 微生物來源之納豆激酶特性比較	39
表4.3 金屬離子對納豆激酶之影響	40
表4.4 TKU015納豆激酶的基質特異性	41
表4.5 TKU015納豆激酶的液相層析串聯式質譜分析	42
表4.6 聚合酶鏈鎖反應實驗之引子	43

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