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研究生:游秋萍
研究生(外文):Chiou-Ping You
論文名稱:構築抗齒舌蘭輪斑病毒之phagedisplaylibrary和篩選單鏈抗體及其親合性分析
論文名稱(外文):Generation and affinities with antigen of single chain variable fragment antibody against Odontoglossum ringspot virus from phage display library
指導教授:顏永福
指導教授(外文):Yung-Fu Yen
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農業生物技術研究所碩士班
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:95
中文關鍵詞:齒舌蘭輪斑病毒單鏈抗體
外文關鍵詞:phage display libraryOdontoglossum ringsport virussingle chain variable fragment antibody
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齒舌蘭輪斑病毒(Odontoglossum ringsport virus,ORSV )是感染蘭科植物重要的病毒,除了影響植株生長外,亦會造成蘭花品質下降,因此,一般用ELISA檢測蘭花ORSV病毒。本研究為構築ORSV phage display library 從中篩選產生scFv 抗體。
本試驗依據ORSV的鞘蛋白(coat protein )基因序列設計引子(primers),將鞘蛋白基因選殖至質體表現系統,由GST(gutathione S-transferase)融合蛋白中純化得到ORSV鞘蛋白,再將鞘蛋白注射至老鼠體內產生免疫反應後,抽取老鼠脾臟細胞的total RNA,進行反轉錄聚合酶連鎖反應(reverse transcription polymerase chain reaction, RT-PCR),將ORSV的抗體重鏈及輕鏈反轉錄成cDNA,利用聚合酵素反應法(polymerase chain reaction,PCR)分別選殖出重鏈及輕鏈的變異區片段,並藉由重疊延伸的基因裁切法(gene splicing by overlap extension)構築出ORSV- scFv DNA,利用phage display system構築ORSV phage display library,再用ORSV鞘蛋白進行親合性的篩選(bio-panning) 三次,篩選到C10、C11、D5、F10等4個重組噬菌體株,這些菌株產生的scFv antibody 對ORSV有專一性的親合反應。以scFv antibody檢查感染ORSV 的蝴蝶蘭葉片萃取液,結果顯示用scFv antibody ELISA 的檢測的OD 405比ORSV多株抗體弱,但最低病毒檢測濃度為10 ng/μl與ORSV 多株抗體的檢測極限相似。本試驗得到的scFv antibody,其檢測靈敏性相當於動物免疫多株抗體,未來希望能利用phage display 技術量產具專一性和靈敏度高的scFv 抗體,應用於病毒的檢測。
Odontoglossum ringspot virus (ORSV) is one of the commonest viral pathogens of the cultivated orchids. Infected plants appear unhealthy and may produce low quality flowers. In this experiment a set of ORSV-specific oligonucleotide primers were designed from the region of the coat protein (CP) gene of ORSV. The ORSV CP gene was cloned into the protein expression bacterial plasmid vector of the gutathione S-transferase(GST)fusion protein expression system. The recombinant ORSV CP was injected into mouse to induce immune response of the animal. The cDNAs of VH and VL of ORSV antibody genes were obtained by using reverse transcription polymerase chain reaction from the total the RNAs that were extracted from the spleen cells of immunized mouse. ScFv(single-chain variable fragment)library of ORSV were constructed with gene splicing by overlap extension. Thirty seven scFvs were selected from ORSV-scFv library following three rounds of affinity selection with ORSV CP as an antigen that was expressed in bacteria. Four scFv antibody have specific binding reaction against ORSV CP was selected. Comparing the sensitivity between scFv antibodies and ORSV polyclonal antibody tisted in enzyme linked immunosorbent assay (ELISA) to detect ORSV in leaf extracts of diseased Phalaenopsis plants. Unfortunately, the affinity between scFv antibody and ORSV was weaker than that between polyclonal antibody to the same antigen. The scFv antibodies and polyclonal antibody appeared to have similar sensitivity limit as low as 10 ng/μl. The results highlight the potential of applying the scFv antibodies in the diagnosis of virus diseases.
目 錄
中文摘要………………………………………………………………………..i
英文摘要……………………………………………………………………….ii
目錄…………………………………………………………………………....iv
圖目次………………………………………………………………………...vii
附錄目次…………………………………………………………………..…...x
壹、前言……………………………………………………………………….……..1
貳、前人研究…………………………………………………….…………….4
參、材料與方法………………………………………………………………15
一、 齒舌蘭輪點病毒鞘蛋白基因片段的選殖
(一) ORSV病毒RNA的純化……………………………………………15
(二)反轉錄聚合酶連鎖反應(Reverse Transcription Polymerase Chain
Reaction, RT-PCR)……………………………………………….…..16
(三) DNA電泳膠體分析……………………………………………….…17
(四) ORSV鞘蛋白基因片段的純化……………………………………...18
(五) ORSV鞘蛋白基因片段之選殖(cloning)…………………………19
(六)定序………………………………………………………………..….22
二、 齒舌蘭輪點病毒鞘蛋白(coat protein)的表現與純化
(一)GST-ORSV 鞘蛋白融合蛋白質的表現……………………………..23
(二)ORSV 鞘蛋白的純化………………………………………….…….23
(三)聚丙醯氨凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel
electrophoresis,SDS- PAGE) …………………………….………..24
(四)西方墨點分析法 (Western blotting)…………………………………25
三、 齒舌蘭輪點病毒(Odontoglossum ringspot tobamovirus, ORSV) scFv
antibody library的構築與表現
(一) ORSV鞘蛋白免疫BALB/cByJ (female)老鼠………………………27
(二)間接酵素連結免疫測定法(indirect enzyme-linked immunosorbent
assay, ELISA )……………………………………………………...27
(三)西方墨點法(western blotting)檢測老鼠產生免疫反應……….….…28
(四)抽取免疫BALB/cByJ(female)老鼠脾臟細胞(spleen cell)的total
RNA………………………………………………………………...28
(五)抗體重鏈/輕鏈PCR放大反應與scFv antibody組裝與表現……….29
四、篩選(biopanning)和soluble scFv 抗體的製備
(一)誘導TG1釋出重組噬菌體…………………………………………...35
(二)scFv 重組噬菌體的篩選(biopanning)…………………………….....35
(三)用ELISA篩選 phage-displayed ORSV scFv抗體………………….38
(四)可溶性抗體片段(soluble scFv antibody)的製備…………………….38
(五)scFv antibody 的純化………………………………………………...40
(六)西方墨點法(Western blot) 分析scFv抗體的表現………………….41
(七)定序分析……………………………………………………………...42
(八)soluble scFv 抗體與蘭花病毒萃取液、ORSV病毒顆粒之間的親合
力檢測……………………………………………………………........42
(九)scFv 抗體濃度定量分析……………………………………………..43
肆、結果………………………………………………………………………44
一、齒舌蘭輪點病毒的選殖…………………………………………………44
二、齒舌蘭輪點病毒鞘蛋白的表現與純化………………………………….49
三、齒舌蘭輪點病毒(Odontoglossum ringspot tobamovirus, ORSV) scFv
DNA library的構築與表現……………………………………………...52
四、scFv 重組噬菌體的篩選(biopanning)與可溶性scFv antibodies的
製備……………………………………………………………………...60
伍、討論……………………………………………………………………….74
陸、參考文獻…………………………………………………………………85
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1. 構築抗喜姆比蘭嵌紋病毒之phagedisplaylibrary和篩選單鏈抗體及其親和性分析
2. 構築對應百合斑紋病毒單鏈抗體及其親和性分析
3. 喜姆比蘭嵌紋病毒單鏈抗體基因轉殖至細菌之研究
4. 抗warfarin之抗體及單鏈抗體之製備、分析與應用
5. 活體外齒舌蘭輪斑病毒台灣系統轉錄體之構築與其5′端鳥糞嘌呤序列數目對於病毒感染力之影響
6. 東亞蘭嵌紋病毒表現載體的構築與分析
7. 東亞蘭嵌紋病毒分離株移動機制的研究與病毒誘導基因靜默系統的建立
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9. 蝴蝶蘭病毒檢測用之核酸快速簡易萃取及黃葉病菌分子鑑定技術之開發
10. 利用噬菌體展現技術製造蘭花病毒鞘蛋白抗原之雞抗體片段重組基因庫
11. 兩種感染蘭科植物重要病毒之檢測法開發及齒舌蘭輪斑病毒感染性選殖株之構築與特性分析
12. 可同時檢測齒舌蘭輪斑及蕙蘭嵌紋病毒之雙特異性抗體之製備與應用
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