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研究生:謝玉貞
研究生(外文):Yu-Chen Hsieh
論文名稱:以含有多個抗原決定位之重組型VP1蛋白為酵素連結免疫吸附分析之披覆抗原偵測口蹄疫病毒抗體
論文名稱(外文):Detection of Antibodies to Foot-and-Mouth Disease Virus by a ELISA Using a Recombinant VP1 Protein with Multi-Epitopes as Coating Antigen
指導教授:鍾楊聰鍾楊聰引用關係
指導教授(外文):Yang-Tsung Chung
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
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:54
中文關鍵詞:口蹄疫病毒酵素連結免疫吸附分析VP1蛋白口蹄疫
外文關鍵詞:Foot-and-Mouth Disease VirusELISAVP1 proteinFoot-and-Mouth Disease
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以含有多個抗原決定位之重組型VP1蛋白為
ELISA披覆抗原 偵測口蹄疫病毒抗體
謝玉貞
國立中興大學獸醫學研究所
口蹄疫病毒(foot-and-mouth disease virus)是不具封套的一種單股RNA病毒,其感染主要對象為豬、牛、羊等偶蹄類動物。本病毒的蛋白衣殼由四種結構蛋白組成,其中VP1已被證實具有兩個可誘發中和抗體的主要抗原決定位(epitopes)。過去他人利用大腸桿菌表現系統所生產之VP1蛋白製成的酵素連結免疫吸附分析(ELISA)檢驗試劑,往往因豬隻血清中含有大量抗大腸桿菌抗體而有測值背景(非特異性結合)過高的嚴重缺點。為了克服此一問題,我運用遺傳工程技術製備含有多個VP1抗原決定位複本的重組蛋白質,以取代傳統式ELISA所使用的單複本蛋白質。我首先以反轉錄酵素-聚合酵素連鎖反應(RT-PCR)方法複製出口蹄疫病毒O型台灣97株的VP1基因片段,然後運用特殊的DNA剪接技巧,分別在pET32a(+)表現載體上嵌入含有一至五個VP1基因複本的DNA片段。在將上述重組質體分別送入大腸桿菌BL21(DE3)菌株中進行表現之後,以鎳離子親和性層析法純化重組基因產物。SDS-PAG分析結果顯示,五種重組蛋白質的分子量一如預期呈現逐級遞增現象。以這些蛋白質為披覆抗原(coating antigen)針對受口蹄疫感染豬隻陽性血清所進行的ELISA測試結果顯示,含有三或四複本數之蛋白質的敏感度較佳,適合開發成為口蹄疫偵測檢驗試劑。

Detection of Antibodies to Foot-and-Mouth Disease Virus
by a ELISA Using a Recombinant VP1 Protein with
Multi-Epitopes as Coating Antigen
Yu-Chen Hsieh
Graduate Institute of Veterinary Medicine, National Chung-
Hsing University
Foot-and-mouth disease virus (FMDV), a positive-stranded RNA virus, is the causative agent of an extremely contagious disease of cloven-hoofed animals, most notably pigs, cattle, and sheep. The capsid of the virion consists of 60 copies of each of the four structural proteins VP1-VP4. Among those, VP1 carries two major B-cell epitopes for inducing neutralizing antibodies. Since the sera of pigs usually contain a large amount of antibodies against Escherichia coli, there is a potential (high non-specific binding) problem in which the E. coli-expressed VP1 protein could be used in a ELISA as coating antigen. To overcome this, I have prepared several kinds of recombinant proteins that have muli-epitopes of VP1 by genetic engineering techniques, instead using single-copy protein as in conventional ELISA. I first amplified the VP1 gene segment of FMDV strain O/TAW/97 by reverse transcriptase-polymerase chain reaction (RT-PCR) method, and then separately incorporate 1 to 5 VP1-copies into the pET32a(+) expression vector. After they were transferred into E. coli cells BL21(DE3) separately, the VP1 gene products were purified by nickel-affinity chromatographic columns. SDS-PAGE showed that the molecular weight of five different recombinant proteins increase stepwise, as expected. The results of the ELISA by using above proteins as coating antigen to test the sera of the FMDV-infected pigs showed that the recombinant VP1 prteins with 3 or 4 copies have better specificity than 1-copy protein.

中文摘要……………………………………………………………… i
英文摘要……………………………………………………………… ii
目次…………………………………………………………………… iii
圖次…………………………………………………………………… vi
中英對照表…………………………………………………………… vii
第1章 緒言………………………………………………………… 1
第2章 文獻探討…………………………………………………… 3
第一節 口蹄疫之歷史背景………………………………………… 3
第二節 口蹄疫病毒的特性………………………………………… 4
2.1 病毒之分類……………………………………………… 4
2.2 病毒之結構及形態學…………………………………… 4
2.3 病毒之血清型…………………………………………… 5
2.4 病毒之基因組成………………………………………… 5
2.5 病毒之理化性質………………………………………… 6
第三節 口蹄疫之病理機制………………………………………… 6
3.1 傳播途徑………………………………………………… 6
3.2 致病機制………………………………………………… 7
3.3 臨床症狀………………………………………………… 7
第四節 免疫反應…………………………………………………… 9
4.1 口蹄疫免疫反應………………………………………… 9
4.2 潛伏感染或帶毒狀態…………………………………… 9
第五節 口蹄疫之診斷……………………………………………… 10
5.1 酵素連結免疫吸附法…………………………………… 11
5.2 核酸鑑識法……………………………………………… 11
第六節 流行病學…………………………………………………… 11
6.1 宿主……………………………………………………… 11
6.2 傳染途徑………………………………………………… 12
6.3 感染動物之排毒及最低感染途徑……………………… 13
6.4 病毒血清型之分布……………………………………… 13
第七節 口蹄疫之預防控制與撲滅策略…………………………… 13
7.1 預防控制………………………………………………… 14
7.2 撲滅策略………………………………………………… 14
第3章 材料與方法………………………………………………… 16
一. 病毒核酸之萃取………………………………………………… 16
二. 反轉錄酵素-聚合酵素連鎖反應 (RT-PCR)…………………… 16
三. RT-PCR 產物之電泳分析與純化……………………………… 17
四. RT-PCR 產物之選殖與核甘酸定序…………………………… 17
五. 重組表現質體之構築…………………………………………… 17
六. 重組基因在大腸桿菌之表現…………………………………… 19
七. 重組基因在大腸桿菌之純化…………………………………… 19
八. 蛋白質的重新摺疊……………………………………………… 20
九. 蛋白濃度的測定………………………………………………… 20
十. 蛋白質之膠片電泳分析………………………………………… 21
十一. 西方免疫轉漬分析…………………………………………… 21
十二. 酵素連結免疫吸附分析 (ELISA)…………………………… 22
第4章 結果………………………………………………………… 24
一. 口蹄疫病毒結構性蛋白VP1基因之選殖與定序……………… 24
二. 含多個VP1基因複本之表現質體的構築……………………… 24
三. 重組蛋白質表現………………………………………………… 25
四. 重組蛋白之純化………………………………………………… 25
五. 表現質體之重構………………………………………………… 26
六. 重構重組蛋白之表現與純化…………………………………… 26
七. 酵素連結免疫吸附分析 (ELISA)……………………………… 26
第5章 討論……………………………………………………… 39
參考文獻……………………………………………………………… 44
附錄…………………………………………………………………… 52

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