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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16943

Title: 巴斯德桿菌磷脂質分解酶訊號胜肽之研究與應用
Studies on phospholipase signal peptide of Photobacterium damselae
Authors: Shuo-Chieh Li
李碩傑
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 訊號胜肽;巴斯德桿菌;表現載體系統;李碩傑;林正輝
signal peptide;Photobacterium damselae;Shuo-Chieh Li;Cheng-Hui Lin
Date: 2008
Issue Date: 2011-06-30T08:45:15Z
Abstract: 摘要 本論文利用signal peptide能將目標蛋白分泌至細胞外的特性,在原核表現系統上利用基因重組的方式來構築新型的表現載體,以期能利用簡單的方式收取蛋白質。 利用化學合成方式合成signal peptide DNA片段,並在兩端設計NdeI/NcoI切位,接入pET32a(+)(NdeI/NcoI)來構築表現載體(p32asp)。將pT2KXIG△in利用限制酶NcoI/NotI將其上的EGFP切割下,並與p32asp(NcoI/NotI)進行接合作用,以構築表現質體p32aspGFP。   將含32aspGFP之菌株以濃度為0.4 mM及1 mM IPTG進行誘導,發現spGFP蛋白在5小時表現量最多,且會在可溶性蛋白表現出28.8 kDa大小的spGFP蛋白,而有少量會表現於不可溶性蛋白中。而在銀染培養液蛋白質可發現,有約26.6 kDa大小的蛋白會在培養液中有表現,大小與GFP蛋白相同。   質體pBScoat與質體p32asp分別以限制酶NcoI/EcoRI切割,並進行接合作用,利用轉形作用將此質體轉入E.coli BL21(DE3),並利用0.4 mM以及1 mM IPTG誘導,可發現spc蛋白表現在1 mM IPTG誘導的可溶性蛋白中有38.8 kDa的大小,而在不可溶性蛋白無表現。而在銀染培養液蛋白質可發現,有約36.6 kDa大小的蛋白會在培養液中有表現。
Abstrat Using the ability of the signal peptide to secrete the target protein out of cell, we try to construct a new expression vector which can express target protein out of cell. DNA of the signal peptide was synthesized by chemical synthesis method. The restriction enzyme sites of NdeI and NcoI were desiged in the end of DNA and ligated with pET32a(+)(NdeI/NcoI) to construct a plasmid called p32asp. EGFP gene digested from pT2KXIG△in by restriction enzyme and ligated with p32asp digested with the same restriction enzyme to construct p32aspGFP. Plasmid p32aspGFP was transformed into E.coli BL21(DE3). The recombinant bacteria were cultured and induce with IPTG. The spGFP protein was expressed in high level after 5 hour induction. The size of spGFP protein is 28.8 kDa in the soluble protein, and 26.6 kDa in culture medium. NNV coat protein gene was digested from pBScoat by restriction enzyme EcoRI and NcoI and ligated with p32asp digested with the same restriction enzyme to construct p32aspc. Plasmid p32aspc was transformed into E.coli BL21(DE3). The recombinant bacteria were cultured and induced with IPTG. The spc protein was expressed the 38.8kDa in 1mM IPTG iduced soluble protein but no expression in insoluble protein. The size 36.6 kDa of spc protein is in culture medium.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95330026
http://ntour.ntou.edu.tw/ir/handle/987654321/16943
Appears in Collections:[水產養殖學系] 博碩士論文

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