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研究生:彭莉雰
研究生(外文):Li-Fen Peng
論文名稱:第一部份:應用DNA重組技術合成ACEI肽第二部份:Lactobacillussake內源性質體之初步探討
論文名稱(外文):Part A: Application of DNA Recombinant Technology to Synthesize ACEI Peptide PartB: Studies on Endogenous Plasmid of Lactobacillus sake
指導教授:李根永李根永引用關係
指導教授(外文):Ken-Yuon Li
學位類別:碩士
校院名稱:東海大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:98
中文關鍵詞:降血壓肽重組蛋白乳酸菌內源性質體
外文關鍵詞:ACEIrecombinant proteinLactobacillus sakeendogenous plasmid
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第一部份:應用DNA重組技術合成ACEI肽
摘 要
本研究是利用DNA重組技術構築大腸桿菌表現系統生產降血壓肽。根據具降血壓功能的RPLKPW(Arg-Pro-Leu-Lys-Pro-Trp)肽,設計兩段互補的降血壓核苷酸序列(ACEI 5 & ACEI 6及ACEI 7 & ACEI 8),利用基因重組技術將這兩段核苷酸序列和表現載體pEZZ18接合在一起,形成能生產降血壓肽的基因載體(pEZZ18-E-RPLKPW),再將此基因載體轉形入宿主細胞E.coli JM105中。篩選出的菌株經液體培養後,利用親和性層析管柱回收具有RPLKPW的融合蛋白(ZZ-E-RPLKPW),進一步將此融合蛋白以V8 protease切割後,利用HPLC分離、純化融合蛋白中的降血壓肽。重組質體經DNA定序儀分析得知降血壓肽基因成功的組合入pEZZ18載體中;帶有重組質體的的轉形菌株所表現的降血壓肽經HPLC分析鑑定,結果與合成的標準品相符合。
第二部份:Lactobacillus sake內源性質體之初步探討
摘 要
本研究從Lactobacillus sake ATCC15521中抽取出一個內源性質體,初步估計其大小為7 kb,命名為pTHU-1。分析序列特性,經限制酶截切反應,可被Hind III、Eco RI、DdeI、AvaII和ScrFI截切,不能被BamH I、Sal I、Xba I、Sca I、Pvu I和NcoI 截切。預測pTHU-1至少有兩個Hind III切點,四個Eco RI切點,2個AvaII切點。Hind III截切得到兩個片段,H1(4kb)和H2(3kb);Eco RI截切得到四個片段,E1(5kb)、E2(1kb)、E3和E4。已定序出E3和E4的序列,E3序列長度為706 bp,E4序列長度為271 bp。E3和E4的序列以BLAST比對沒有相符合的質體,各自比對找出四個orf,也沒有相符合的基因。
PartA: Application of DNA Recombinant Technology to Synthesize ACEI Peptide
Abstract
In this study, DNA recombinant technique was performed to construct a prokaryotic expression vector which carrys angiotensin I-converting enzyme inhibitor peptide (ACEI). The recombinant vector confers on the transformed host cell the ability to produce ACEI peptide. Two complementary polynucleotides encoding ACEI peptide (ACEI 5&6 and ACEI 7&8) were synthesized. The double strand polynucleotides with Sal I sticky end were inserted into pEZZ18, and then the recombinant plasmid (pEZZ18-E-RPLKPW) were transformed to E.coli JM105. Some transformed E.coli colonies with the ability to produced peptide were isolated. The DNA sequence of ACEI peptide inserted into pEZZ18 was analyzed successfully by DNA sequencer. The peptide linked with ZZ protein was isolated with an affinity chromatography. The isolated fusion protein was treated with V8 protease to release the peptide. The ACEI peptide was analyzed with an HPLC system.
PartB: Studies on Endogenous Plasmid of Lactobacillus sake
Abstract
A endogenous plasmid was isolated from Lactobacillus sake ATCC15521. The size of this plasmid was determined as about 7-kb. Two Hind III and Eco RI restriction sites were found on the plasmid. H1 and H2 fragments were generated by Hind III with molecular size about 4-kb and 3-kb, respectively. E1(5-kb), E2(1-kb), E3(706 bp), and E4(271 bp) were the fragments generated by using Eco RI. The small fragments, E3 and E4, were sequenced. No identified sequence were found in the database of BLAST. The sequences of the fragments possess eight ORFs but no gene function were identified yet. The novel endogenous plasmid was named pTHU-1.
目 錄
第一部份:應用DNA重組技術合成ACEI肽
中文摘要-------------------------------------------------I
英文摘要-------------------------------------------------II
壹、前言-------------------------------------------------1
貳、文獻回顧---------------------------------------------2
一、血管收縮素與高血壓的關係--------------------------2
二、血管收縮素轉換酶----------------------------------4
三、血管收縮素轉換酶抑制劑----------------------------6
四、食品中抗高血壓肽發展現況------------------------7
五、RPLKPW的發現--------------------------------------9
六、應用DNA重組技術合成---------------------------11
參、材料與方法-------------------------------------------15
一、材料-------------------------------------------------15
(一)菌種及表現載體---------------------------------15
(二)培養基及篩選培養基-----------------------------15
(三)儀器設備---------------------------------------17
二、方法-------------------------------------------------18
(一) 構築pEZZ18-E-RPLKPW---------------------------18
1.表現載體pEZZ18的製備-----------------------------18
1.1抽取質體---------------------------------------18
1.2電泳分析--------------------------------------19
1.3限制酶截切反應--------------------------------20
1.4純化限制酶截切後之pEZZ18(Sal I)---------------20
2.RPLKPW 序列之製備--------------------------------20
2.1設計及合成序列-----------------------------------20
2.2 粘合作用(annealing)--------------------------21
2.3 限制酶截切反應---------------------------------21
2.4 純化E-RPLKPW基因片段---------------------------22
3.接合作用(Ligation)-------------------------------------22
4 勝任細胞(competent cell)及轉形作用(transformation)-----23
4.1勝任細胞(competent cell)-----------------------23
4.2轉形作用(transformation)-----------------------24
5. 確定成功轉型株----------------------------------------24
6. 定序反應----------------------------------------------24
(二) 分離及純化RPLKPW------------------------------------25
1.發酵轉型菌株--------------------------------------25
2. 親和性管柱層析-----------------------------------25
3. 蛋白質濃度測定----------------------------------------27
4. 利用V8 protease水解ZZ-E-RPLKPW融合蛋白----------------27
5. HPLC 分析--------------------------------------------27
6. SDS-聚丙醯胺膠體電泳分析------------------------------28
6.1 配製方法-----------------------------------------28
6.2 染色-------------------------------------------------31
肆、結果與討論-------------------------------------------32
一、將RPLKPW基因選殖至大腸桿菌系統-----------------------32
(一)製備降血壓肽核苷酸序列-----------------------32
(二)轉形菌的篩選-----------------------------------34
二、融合蛋白的分離---------------------------------------35
三、RPLKPW肽之HPLC分析---------------------------------36
伍、結論-------------------------------------------------37
陸、參考文獻---------------------------------------------51
附錄一---------------------------------------------------55
附錄二---------------------------------------------------57
第二部份:Lactobacillus sake內源性質體之初步探討
中文摘要-------------------------------------------------III
英文摘要-------------------------------------------------IV
壹、前言-------------------------------------------------58
貳、文獻回顧---------------------------------------------59
一、乳酸菌選殖載體---------------------------------------59
二、食品級載體表達系統--------------------------------61
(一)食品級篩選標制系統------------------------------61
(二)食品級染色體插入系統----------------------------63
(三)食品級調控表現系統------------------------------63
三、 L. sake宿主選殖系統---------------------------------64
(一) L. sake之基本性質------------------------------64
(二) L. sake在基因工程上的發展----------------------69
參、材料與方法-------------------------------------------71
一、材料----------------------------------------------71
(一)菌株-------------------------------------------------71
(二)構築載體---------------------------------------------71
(三)培養基-----------------------------------------------71
(四)儀器-------------------------------------------------71
二、方法-------------------------------------------------72
1. L. sake的培養與保存-----------------------------72
2.小量抽取質體-------------------------------------72
3.大量抽取質體--------------------------------------73
4.DNA電泳-------------------------------------------74
5.限制酶截切反應------------------------------------74
6.膠上純化基因片段----------------------------------75
7.構築----------------------------------------------75
8.定序----------------------------------------------76
9.序列比對------------------------------------------76
肆、結果與討論-------------------------------------------77
一、內源性質體的抽取----------------------------------77
二、L. sake內源性質體限制酶作用位置-------------------79
三、將限制酶截切片段部分定序--------------------------80
伍、結論-------------------------------------------------83
陸、參考文獻---------------------------------------------94
Part A: Application of DNA Recombinant Technology to Synthesize ACEI Peptide
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PartB: Studies on Endogenous Plasmid of Lactobacillus sake
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