Pseudomonas sp. TKU015殺蟲蛋白之基因選殖與純化

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Title:	Pseudomonas sp. TKU015殺蟲蛋白之基因選殖與純化
Other Titles:	Molecular cloning and purification of an insecticidal protein from pseudomonas sp. TKU015
Authors:	林詠迪;Lin, Yung-di
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	Pseudomonas sp.;納豆激酶;基因選殖;殺蟲蛋白;Pseudomonas sp.;nattokinase;gene cloning;insecticidal protein
Date:	2009
Issue Date:	2010-01-11 02:27:58 (UTC+8)
Abstract:	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.
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