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研究生:李碩傑
研究生(外文):Shuo-Chieh Li
論文名稱:巴斯德桿菌磷脂質分解?訊號胜?之研究與應用
論文名稱(外文):Studies on phospholipase signal peptide of Photobacterium damselae
指導教授:林正輝林正輝引用關係
指導教授(外文):Cheng-Hui Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:71
中文關鍵詞:訊號胜?巴斯德桿菌表現載體系統李碩傑林正輝
外文關鍵詞:signal peptidePhotobacterium damselaeShuo-Chieh LiCheng-Hui Lin
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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?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?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.
目錄

摘要
壹、前言……………………………………………………………… …1
一、基因表現載體系統之簡介………………………………………… 1
1、動物細胞系統…………………………………………………………1
高等動物細胞系統………………………………………………1
低等動物細胞系統………………………………………………2
植物細胞系統 ………………………………………………… 4
2、微生物細胞系統………………………………………………………5
真核微生物細胞系統……………………………………………5
原核微生物細胞系統 ………………………………………… 6
二、巴斯德桿菌概述 …………………………………………………… 8
三、訊號胜?序列之簡介……………………………………………… 10
蛋白質的製造過程及路徑………………………………… 10
訊號胜?簡介……………………………………………… 11
四、研究目的………………………………………………………… 14
貳、材料方法………………………………………………………… 15
一、構築實驗表現載體p32asp……………………………………… 15
1、合成訊號胜?序列(sp)……………………………………………15
2、p32asp的構築 ……………………………………………………… 15
pET-32a(+)載體之製備………………………………………15
接合作用(Ligation) ………………………………………… 16
勝任細胞(competent cell)製備……………………………… 16
轉形作用(transformation)…………………………………… 17
  微量質體DNA的純化(Mini-preparation of plasmid DNA)...18
質體DNA限制?切割(Restriction digestion of plasmid DNA) 確認 …………………………………………………………… 18
目標基因定序 ………………………………………………… 19
二、p32aspGFP質體之構築 ……………………………………………19
1. p32aspGFP構築 ……………………………………………………19
2. 重組spGFP之表現 ………………………………………………… 20
重組spGFP之誘導 ………………………………………… 20
蛋白質之製備 ……………………………………………… 20
細胞外蛋白質收取………………………………………… 20可溶性與不可溶性蛋白收取 …………………………………21
SDS-聚丙烯醯膠蛋白質電泳(SDS-PAGE)…………………22
三、構築p32aspcoat質體 …………………………………………… 22
1. pBScoat質體構築 ………………………………………………… 22
pBluescript II SK(+)載體之製備 …………………………… 22
利用聚合?連鎖反應(polymerase chain reaction,PCR)選殖coat protein基因 ………………………………………………… 23
純化coat protein PCR產物 ………………………………… 24
製備pBScoat質體……………………………………………… 24
2. p32aspc質體製備…………………………………………………… 24
3.重組coat protein誘導及製備 ……………………………………… 25
4.硝酸銀(Ammoniacal Silver)染色………………………………… 26
參、結果…………………………………………………………………27
一、p32asp表現載體之構築 ……………………………………………27
二、p32aspGFP質體之構築 ……………………………………………27
三、p32aspGFP蛋白質表現 ……………………………………………27
四、pBScoat質體之構築 ……………………………………………… 28
五、p32aspc質體之構築 ……………………………………………… 28
六、p32aspc蛋白質表現 ……………………………………………… 29
肆、討論 …………………………………………………………………30
一、signal peptide特性探討 …………………………………………… 30
二、p32aspGFP表現分析 ……………………………………………… 31
三、p32aspc表現分析 ………………………………………………… 32
伍、參考文獻 …………………………………………………………33
陸、圖表 …………………………………………………………………42
伍、參考文獻

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