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研究生:周怡廷
研究生(外文):Yi-Ting Chou
論文名稱:以菌體表層展示技術表現重組之表層蛋白及融合蛋白於大腸桿菌、乳酸桿菌及乳酸鏈球菌
論文名稱(外文):Expression of recombinant S-layer and fusion proteins in Escherichia coli, Lactobacillus paracasei and Lactococcus lactis by cell surface display technology
指導教授:葉娟美
指導教授(外文):Chuan-Mei Yeh
學位類別:碩士
校院名稱:國立中興大學
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
中文關鍵詞:乳酸菌表層蛋白質腸病毒71型外鞘蛋白訊息胜肽
外文關鍵詞:Lactic acid bacteriaSurface layer proteinEnterovirus 71-VP1Signal peptide
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中文摘要
表層(surface layers, S-layers)主要是由單分子的蛋白質或是醣蛋白質所組成,稱為表層蛋白質(surface layer protein,S-protein),會在細胞表面形成孔狀結構。S-layers具有維持細胞形態、做為酵素的骨架及細胞附著和感染等功能,至目前為止尚未有足夠的研究證明其結構與功能間的相關性。有些乳酸菌具有S-protein,例如Lactobacillus acidophilus ATCC 4356,S-protein在乳酸桿菌屬的菌體中所扮演的的角色目前仍未清楚,但在細胞附著上扮演著重要的角色。腸病毒71型VP1是腸病毒的外鞘蛋白質,在本實驗中,與表層蛋白質N端融合表現。
本實驗主要的目的為利用細胞表層展示技術展現重組蛋白於食品級菌株之細胞表面。本實驗選用具有細胞附著功能的S-protein C端123個胺基酸做為研究,利用聚合酶連鎖反應合成出其相對應的DNA序列,並與腸病毒外鞘蛋白基因進行融合,將融合後的基因選殖於不同質體中,並在大腸桿菌、乳酸桿菌及乳酸練球菌中表現。大腸桿菌中,pET表現系統於IPTG誘導後,可以表現重組表層蛋白,此蛋白經純化後,已用來進行抗體之製備。在乳酸桿菌中所表現之融合蛋白,經由細胞劃分後,發現均表現在細胞壁、細胞膜與細胞質及分泌於胞外。在乳酸鏈球菌中表現之融合蛋白大都表現在細胞壁、細胞膜與細胞質,有極微量分泌到胞外。本實驗結果顯示,乳酸鏈球菌是表現外鞘及表層融合蛋白較合適的宿主,不過在蛋白質的表現量應該可以再進一步改進。本研究結果可用來開發口服乳酸菌疫苗,發揮腸道免疫系統。
Abstract
Surface layers (S-layers) protein are crystalline monomolecular composed of single protein or glycoprotein, which formed porous lattices covering the cell surface. It’s biological function includes maintenance of cell shape, cell adhesion, molecular sieve, and scaffolding for enzyme. Some lactic acid bacteria (LAB) such as Lactobacillus acidophilus ATCC4356, contain S-protein, which may play an important role in cell adhesion. Enterovirus 71 VP1(EV71VP1) is a capsid protein of enterovirus, in this study, we fused this protein to the C-terminal 123 a.a. of S-protein.
This research was aimed to express the recombinant protein on the food grade bacteria cell surface by cell surface display technology. The DNA sequence of S-protein C-terminal 123a.a. (Sbc) with cell adhesion function was synthesized by polymerase chain reaction and fused to the capsid protein gene of enterovirus. The function gene was then cloned into the expression vector and transformed into Escherichia coli, Lactobacillus paracasei and Lactococcus lactis by electroporation. In E. coli, the recombinant S-protein C-terminal 123a.a. (rSbc) was expressed using the pET expression system through isopropyl-β-D- thiogalactopyranoside (IPTG) induction. rSbc was purified and used to prepare anti-Sbc antibody. In L. paracasei, rSbc and fusion protein were expressed on cell wall, cell membrane, cytosol and secreted into medium. In L. lactis, rSbc and fusion protein were expressed on cell wall, cell membrane and cytosol but few into medium. Results showed that the synthesized Sbc and fused EV71VP1 gene could express in all the hosts tested. The L. lactis is a more preferable host for Sbc cell surface display.
內容目錄 頁次
中文摘要 ……………………………………………………….…. i
英文摘要 …………………………………………………….……. ii
壹、前言 …………………………………………………….……. 1
貳、實驗目的 ……………………………………………….……. 20
參、實驗策略……………………………………………………… 20
肆、材料與方法 ………………………….………………………. 24
一、菌種與質體………………………………………………….... 24
二、藥品與試劑…………………………………………………… 26
三、質體抽取法…………………………………………………… 27
四、DNA分子之電泳、剪切、回收及黏合…………………….. 28
五、電轉形………………………………………………………… 29
六、表現元件基因之增幅 …………..…………………………… 32
七、大腸桿菌與乳酸菌表現載體之建構………………………… 35
八、表層蛋白基因於大腸桿菌之表現偵測……………………… 38
九、表層蛋白基因於乳酸桿菌之表現偵測……………………… 38
十、表層蛋白基因於乳酸鏈球菌之表現偵測…………………….. 38十一、融合蛋白基因於乳酸桿菌之表現偵測…………………… 39
十二、融合蛋白基因於乳酸鏈球菌之表現偵測………………… 39
十三、大腸桿菌來源的表層蛋白質樣品之製備………………… 40
十四、乳酸菌來源之表層蛋白質樣品之製備…………………… 40
十五、Ni-NTA金屬螯合樹脂親和性層析法純化蛋白質……….. 41
十六、表層蛋白抗體之製備…………………………………….... 41
十七、表層蛋白抗體之純化……………………………………… 42
十八、表層蛋白特性分析………………….……………………… 42
十九、融合蛋白特性分析………………….……………………… 43伍、結果與討論…………………………………………………… 45
一、表層蛋白之合成………………………….…………………… 45
二、腸病毒71型外鞘蛋白VP1(EV71VP1)之合成……………… 45
三、大腸桿菌與乳酸菌表現載體之構築………………………… 49
四、表層蛋白基因在大腸桿菌中表現情形….…………………… 68
五、蛋白質在乳酸桿菌中表現情形……………………….……… 68
六、蛋白質在乳酸鏈球菌中表現情形…………………………… 73
陸、結論 ………………………………………………………….. 81
柒、參考文獻 …………………………………………………….. 82
柒、參考文獻
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