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研究生:張超銘
論文名稱:乳酸球菌Lactococcuslactis無質體菌株的分離,並以其選殖載體銜接Epstein-Barr病毒醣蛋白gp25基因
論文名稱(外文):Isolation of plasmid-free strain in Lactococcus lactis, and Cloning Epstein-Barr virus (EBV) gp25 gene with L.lactis cloning vector
指導教授:林志侯
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:55
中文關鍵詞:乳酸球菌選殖載體無質體菌株電穿孔穿梭載體
外文關鍵詞:Lactococcus lactiscloning vectorplasmid-free strainelectroporationshutter vector
相關次數:
  • 被引用被引用:3
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乳酸菌為革蘭氏陽性菌,一般被視為是安全性的菌種(generally
regarded as safe; GRAS)。以乳酸菌表現異質蛋白質和承載一段抗原基因形成融合蛋白,是一個極具潛力的抗原傳送媒介。
我們發展了一套乳酸球菌Lactococcus lactis的分泌表現分析系統。Staphylococcal nuclease(Nuc)是一種可以被乳酸菌分泌出細胞外的小蛋白質,其具有穩定良好的生化特徵,可以當作乳酸菌融合表現異質蛋白的報導基因(reporter gene)。將乳酸球菌選殖載體pNuc10上的Nuc蛋白N-端分泌訊號後方17個殘基切除 (形成NucT蛋白) ,會減少分泌量至30%,若是在NucT蛋白N-端分泌訊號後方再插接一段帶有負電荷的合成peptide LEISSTCDA,則會使得分泌量上升至90%,如此可以提高Nuc異質蛋白的產量。
由於乳酸球菌選殖載體pNuc10並無法在E.coli的細胞中複製,故我們以E.coli的載體pBluescriptⅡSK(+)融合銜接pNuc10以形成一穿梭載體(shutter vector)pBN,經熱休克(heat shock)方式將DNA先轉形至E.coli,藉由ColE1複製成超螺旋結構,再電穿孔(electroporation)至乳酸球菌L.lactis MG1363中進行表現。
實驗中欲表現的抗原為EB 病毒外殼醣蛋白gp25,將其接上六個組氨酸(histidine)的標誌蛋白片段,並一起銜接到NucT蛋白與LEISSTCDA之間,使其形成融合蛋白,期望藉由乳酸菌進入動物腸道中分泌表現出來,以引發腸黏膜的免疫反應,達到發展活菌疫苗的效果。
Lactic acid bacteria (LAB) are Gram-positive bacteria and generally regarded as safe (GRAS) organisms. LAB could be used for heterologous protein secretion and fused antigen to form fusion protein. They are good potential candidates as antigen delivery vehicles. We developed an efficient secretion system in the Lactococcus lactis model. Staphylococcal nuclease (Nuc protein) is a small, stable, and biochemically well-characterized enzyme secreted by Gram-positive bacteria. It was used as the reporter protein. Plasmid pNuc10 encoding
LEISSTCDA-NucT is a L. lactis cloning vector. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). A 9-residue synthetic propeptide, LEISSTCDA , was fused immediately after the signal peptide cleavage site. Secretion efficiency was increased to ~90% by LEISSTCDA insertion without altering the signal peptide cleavage site.
Because the cloning vector, pNuc10, could not replicate in E.coli. We fused pBluescriptⅡSK(+) and pNuc10 to form the shutter vector pBN. We transformed DNA to E.coli by heat shock, and it would replicate in supercoil form for ColE1 origin of E.coli. Then the expression vector is transferred to the L. lactis MG1363 strain by electroporation. In this study, the gp25 glycoprotein of EB virus was fused with histidine tag as a heterologous protein, and was cloned between NucT and LEISSTCDA to form fusion protein.
We hope that the L.lactis MG1363 transformed with this expression vector can be used as a live vaccine system.
英文摘要………………………………………………………………Ⅰ
中文摘要………………………………………………………………Ⅱ
縮寫對照表……………………………………………………………Ⅵ
前言 ……………………………………………………………………1
材料及方法 ……………………………………………………………9
1.材料…………………………………………………………………9
1.1細菌菌株…………………………………………………………………9
1.2質體 ……………………………………………………………10
1.3引子 ……………………………………………………………10
1.4試劑 ……………………………………………………………10
2.方法 ……………………………………………………………………………11
2.1 細菌的養殖及保存…………………………………………………………..11
2.2從細菌中抽取少量DNA…………………………………………………….11
2.2.1 E.coli質體DNA的抽取(微量) ………………………………………11
2.2.2 Lactococcus 和Lactobacillus質體DNA的抽取(微量)……………….12
2.3 E.coli質體DNA的抽取(中量)………………………………………………13
2.4 Lactococcus 染色體DNA的抽取 ………………………………………….14
2.5聚合酶連鎖反應 ……………………………………………………………..15
2.6 DNA片段回收法 ……………………………………………………………15
2.7 連接反應 …………………………………………………………………….16
2.8轉形細胞 (勝任細胞) 之製備 ……………………………………………...16
2.9細菌轉形作用 ………………………………………………………………..17
2.9.1 熱休克法…………………………………………………………………..17
2.9.2電穿孔法…………………………………………………………………...17
2.10南方雜配……………………………………………………………………..18
2.10.1放射性標定探針的製備 ………………………………………………...19
2.10.2南方轉漬及雜配反應 …………………………………………………...19
2.11 SDS 聚丙烯醯胺膠電泳法分析……………………………………………21
結果……………………………………………………………………23
1.乳酸球菌選殖載體pNuc10上NucT報導基因的表現測試…………………...23
2.乳酸球菌選殖載體pNuc10的map……………………………………………..23
3.對L.lactis MG1363(含pNuc10質體)進行質體剔除和尋找pNuc10選殖載體
的宿主………………………………………………………………………………23
3.1對L.lactis MG1363(含pNuc10質體)進行質體剔除………………………...24
3.2尋找pNuc10選殖載體的宿主………………………………………………..24
4.測定L.lactis MG1363的生長曲線………………………………………………25
5.建構穿梭載體pBN……………………………………………………………….25
6.以PCR合成EB病毒gp25基因………………………………………………...26
7. pBN穿梭載體的測試:pGBN載體的建構……………………………………...27
8.以pGBN表現gp25蛋白的測試……………………………………………………28
討論……………………………………………………………………..30
參考資料………………………………………………………………..34
圖表……………………………………………………………………..38
附錄……………………………………………………………………..52
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