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研究生:張致維
研究生(外文):chih-wei chang
論文名稱:利用家禽生產抗腸病毒71型之蛋黃免疫球蛋白
論文名稱(外文):Egg yolk immunoglobulin (IgY) against enterovirus 71
指導教授:戴謙戴謙引用關係劉瑞珍黎煥耀黎煥耀引用關係
指導教授(外文):Chein TaiJui-Jane LiuHuan-Yao Lei
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2005
畢業學年度:93
語文別:中文
論文頁數:91
中文關鍵詞:免疫球蛋白腸病毒71型
外文關鍵詞:IgYEV71
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  近年來家禽蛋黃所生產之免疫球蛋白(immunoglobulin in yolk, IgY)頗為受到重視,主要在於IgY含有生產方式較經濟、純化方法簡易、抗體產量高等多項優點。目前IgY已被嘗試用來提供大腸桿菌、沙門氏桿菌、幽門桿菌、輪狀病毒等多種病原之抗體,都顯示可以有效減輕病原之效果。腸病毒71型會引發腦膜炎、小兒麻痺等症狀,容易感染5歲以下幼兒,由於1998年在台灣引起大流行而受到極大重視,近幾年仍有一些個別案例存在,因此本研究希望能利用IgY作為腸病毒治療用之抗體來源,並探討其應用於預防及治療之效果。首先,以不活化之腸病毒71型作為抗原,免疫來亨雞生產含anti-EV71 IgY的免疫雞蛋,之後以水溶液稀釋法將IgY從蛋黃中分離。每週蛋黃經純化後所獲得的水溶性部分(water soluble fraction,WSF),經由酵素免疫分析法(ELISA)測得結果,第3週起anti-EV71 IgY含量漸升高,在免疫後第8週平均特異性抗體含量最高,平均特異性IgY約佔IgY的1.3%,在改良後的2005批次免疫效果更佳,在免疫後第6週即可發現anti-EV71 IgY含量可提高至IgY的3%。將生產的anti-EV71 IgY濃縮液進行腸病毒71型的in vitro中和試驗,發現濃度為17~21μg/ml以上anti-EV71 IgY可對100TCID50之腸病毒71型有中和效果,使病毒無法感染人類橫紋肌肉瘤細胞(RD)而達到保護效果,所純化的IgY批次中,最高的病毒中和力價可達到512。在ICR小鼠的腸病毒動物模式實驗中,以1天大小鼠進行腹腔注射(IP)攻毒,隔天以力價256以上的IgY濃縮液給予IP治療,發現存活率可提高至100%,而對照組存活率為20%以下,若在第三天才給予力價512的IgY濃縮液,仍可達到79%存活率。在口服IgY模式測試實驗中,以力價512濃縮IgY液體,採口服管餵食方式,在攻毒前或攻毒後1小時進行治療,初步結果發現對小鼠有顯著保護效果,日後可對於給予時間及劑量上所造成的治療效果作進一步探討。期待未來IgY能以針劑或食品方式用於腸病毒71型的治療及預防。
 The immunoglobulins from poultry egg yolk (IgY) have been evaluated in the reagents of diagnostics and therapy of gastrointestinal diseases in recent years. The IgY products, other sources of polyclonal antibodies, have been proved to be more economic, easier in purification steps, higher yields, and non-invasive to animals. It has been tried in providing the IgY as therapeutic antibodies, for Escherichia coli; Salmonella; Helicobacter pylori; Rotavirus and so on, and they were all effective.

 This study aims at producing IgY against Enterovirus 71, which has been widespread in 1998 and caused the death of 78 children in Taiwan. It will cause the syndromes of diarrhea, foot and mouth disease, aseptic meningitis and poliomyelitis in the infection of children below 5 years old. In this study, we use inactivated EV71 cell lysate as antigen, and immunized Leghorn hens six times at an interval of two weeks to produce eggs with anti-EV71 IgY. After using simple water dilution method to separate IgY from yolk, we determine the quantity of anti-EV71 IgY in the water soluble fraction (WSF) by ELISA. The concentration of specific antibody against EV 71 rose from third week after first immunization, and reached the highest response at 8th week. The WSF contained 1.3% anti-EV71 specific IgY of total IgY at 8th week. The batch of 2005 IgY contained 3% anti-EV71 specific IgY at 6th week after modifying the immunization protocols.

 In vitro EV71 neutralization tests were conducted by the infection of EV 71 on human rhabdomyosarcoma cell line (RD) after adding the purified IgY. The results indicated the concentration higher than 17-21μg/ml anti-EV71 IgY was able to neutralize 100TCID50 EV71and protect RD cells from infection. The highest neutralization titer (NT) obtained in this study was 512. We also evaluated the in vivo protection effect of anti-EV71 IgY on ICR mice model. One day old ICR mice were challenged with EV 71 strain MP4 intraperitoneally (IP), and purified IgY was administrated by IP injection on following day for 3 consecutive days after the viral challenge. It showed antibody with the NT value higher than 256 was capable of protecting the challenged mice with 100% survival rate, while the control group had a survival rate below 20%. If the anti-EV71 IgY of 512 neutralization titer was injected from third day after viral challenge, a 79% survival rate was obtained. The oral administration of 512 IgY (NT512) also showed significant protection in a preliminary test, more studies needs to be done to clarify the effect of IgY titer and administration time in oral tests.

 However, the effective therapeutic function by IP injection in mouse EV 71 animal model suggests the highly potential application of anti-EV71 IgY in the human EV71 infection, with more studies on the development of IgY powder for preventive objective, or by highly purified IgY as the IP injection solution.
中文摘要.....................................I
Abstract.....................................II
誌謝.........................................IV
目錄.........................................VI
圖目錄.......................................IX
表目錄........................................X
縮寫檢索表...................................XI
前言..........................................1
文獻檢討......................................3
一、被動免疫治療..............................3
二、家禽雞蛋免疫球蛋白(IgY)簡介...............4
1 雞蛋組成成分及IgY來源.....................4
2 IgY和IgG的異同...........................5
3 IgY作為抗體來源的優點....................6
4 IgY的分離純化............................9
三、家禽雞蛋免疫球蛋白(IgY)的應用.............9
1 診斷應用.................................9
2 被動免疫治療............................10
3 Gene-specific IgY之應用.................11
四、腸病毒概論...............................13
五、腸病毒71型...............................16
1 病毒結構與性質..........................16
2 病毒感染與症狀..........................16
3 病毒流行歷史與檢測方式..................18
六、腸病毒71型治療及預防.....................19
1 Antiviral agents....................19
2 Vaccine.............................20
3 Personal health care....................21
材料與方法...................................22
一、試驗流程圖...............................22
二、試驗材料與方法...........................23
1. 家禽免疫................................23
1.1 家禽來源........................23
1.2 免疫之抗原......................23
1.3 免疫步驟........................23
1.4 免疫雞蛋之收集..................23
2 IgY分離與純化.......................24
2.1 水溶液分離法....................24
2.2 進階IgY純化.....................24
3 蛋黃中總蛋白、總IgY、anti-EV71 IgY定量分析25
3.1 總蛋白分析.....................25
3.2 總IgY定量......................25
3.3 Anti-EV71 IgY定量..............26
4 In vitro neutralization test of IgY.......27
4.1 RD細胞培養..............................27
4.2 EV71病毒培養............................28
4.3 Anti-EV71 IgY病毒中和測試...............30
5 In vivo protection test of IgY............32
結果.........................................34
一、IgY純化液(WSF)蛋白及抗體含量分析.........34
二、Anti-EV71 IgY in vitro病毒中和測試.......35
三、in vivo ICR mice protection test.........36
1 腸病毒感染症狀及劑量探討................36
2 IgY被動免疫治療效果.....................36
討論.........................................38
一、IgY純化液(WSF)蛋白及抗體含量分析.........38
二、Anti-EV71 IgY in vitro病毒中和測試.......39
三、in vivo ICR mice protection test ........40
結論.........................................44
結果圖表.....................................45
參考文獻.....................................63
自述.........................................79
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