腺病毒於禽流感M2疫苗發展之評估

Shu-Ping Lin; 林淑萍

請用此 Handle URI 來引用此文件： http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65214

完整後設資料紀錄

DC 欄位	值	語言
dc.contributor.advisor	王金和(Ching-Ho Wang)
dc.contributor.author	Shu-Ping Lin	en
dc.contributor.author	林淑萍	zh_TW
dc.date.accessioned	2021-06-16T23:30:14Z	-
dc.date.available	2017-08-01
dc.date.copyright	2012-08-01
dc.date.issued	2012
dc.date.submitted	2012-07-28
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dc.identifier.uri	http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65214	-
dc.description.abstract	一般商業化之家禽流行性感冒病毒之疫苗對於不同HA亞型之家禽流行性感冒病毒感染無交叉保護力。因此，本實驗以發展具交叉保護之疫苗為目的，利用複製缺陷腺病毒 (replication deficient adenovirus) 作為載體，建構帶有不同亞型間保守度高之M2基因片段的重組腺病毒 (Ad/M2)，再將Ad/M2免疫雞隻。Ad/M2感染之293A細胞及轉導之Vero細胞以間接螢光免疫分析皆有陽性訊號產生。在Ad/M2免疫雞隻的實驗結果顯示，補強後2週CD4+及CD8+ T cell相較於初次免疫有顯著增加；抗M2特異性抗體則於初次免疫後2週有顯著增加，但補強後的抗體上升效果則不明顯。於攻毒後第4天及第7天採集的喉頭拭子之real time RT-PCR結果顯示免疫組與對照組之間的AIV含量沒有差異，而攻毒後免疫組與對照組死亡率分別為40%及33.3%，給予Ad/M2之組別並無法對抗AIV感染。	zh_TW
dc.description.abstract	Current vaccines to avian influenza virus can’t induce broad cross-protection against different subtypes. Therefore, the purpose of this research was to develop a vaccine which can confer cross-protection by using replication-deficient adenovirus as a vector to construct a recombinant adenovirus carrying highly conserved M2 gene (Ad/M2). The immunofluorescence assay revealed positive signal in 293A and Vero cells transduced with Ad/M2. The Ad/M2 was intranasally and intramuscularly immunized in chickens twice at a 4-week interval. Anti-M2 specific antibody was significantly higher after two weeks post primary vaccination. Challenging with AIV after two weeks post boost vaccination, the consequence of real time RT-PCR revealed AIV content was no difference between Ad/M2 group and control group in oropharyngeal swab on day 4 and day 7 post challenge. The mortality of Ad/M2 group and control group was 40% and 33.3% respectively. The conclusion is giving chickens Ad/M2 couldn’t confer protection against AIV infection.	en
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dc.description.tableofcontents	目錄口試委員審定書…………………………………………………………………………i 致謝……………………………………………………………………………………………ii 摘要…………………………………………………………………………………………iii Abstract………………………………………………………………………………iv 目錄……………………………………………………………………………………………v 表目錄…………………………………………………………………………………viii 圖目錄………………………………………………………………………………………ix 第一章序言…………………………………………………………………………………1 第二章文獻回顧…………………………………………………………………………3 第一節病毒病原學……………………………………………………………………3 2-1.1 病毒分類……………………………………………………………3 2-1.2 病毒命名……………………………………………………………3 2-1.3 病毒結構與特徵………………………………………………4 2-1.4 物理化學特性…………………………………………………11 2-1.5 病毒複製機制…………………………………………………11 2-1.6 病毒病原性……………………………………………………12 2-1.7 病毒變異性……………………………………………………13 第二節病毒流行病學………………………………………………………………14 2-2.1 宿主範圍……………………………………………………………………14 2-2.2 病毒傳播……………………………………………………………………15 2-2.3 臨床症狀與病理變化………………………………………………15 2-2.4宿主免疫反應……………………………………………………………16 2-2.5預防與控制…………………………………………………………………17 第三節腺病毒…………………………………………………………………………19 2-3.1腺病毒結構與特性……………………………………………………19 2-3.2 宿主免疫反應…………………………………………………………19 2-3.3 複製缺陷之腺病毒載體…………………………………………20 第三章材料與方法…………………………………………………………………23 第一節 M2基因選殖………………………………………………………………23 3-1.1 病毒來源…………………………………………………………………23 3-1.2 病毒核酸檢測…………………………………………………………23 3-1.3 M2基因選殖……………………………………………………………24 3-1.4 pENTR-M2之建構…………………………………………………27 3-1.5 pENTR-M2之轉型…………………………………………………28 第二節 pAd-M2 plasmid之建構……………………………………30 3-2.1 重組反應…………………………………………………………………30 3-2.2 pAd-M2 plasmid之轉型……………………………………30 3-2.3 Pac I限制酶酵素作用……………………………………………31 第三節 Ad/M2之製備………………………………………………………………32 3-3.1 293A細胞之培養………………………………………………………32 3-3.2 pAd-M2 plasmid轉染作用…………………………………32 3-3.3 Ad/M2之增殖與病毒力價提升………………………………33 3-3.4 Ad/M2力價測定…………………………………………………………33 第四節 Ad/M2之M2蛋白表現…………………………………………………34 3-4.1 用於表現M2之細胞株………………………………………………34 3-4.2 M2蛋白之偵測……………………………………………………………34 第五節 Ad/M2之免疫流程………………………………………………………36 3-5.1 實驗動物………………………………………………………………………36 3-5.2 免疫………………………………………………………………………………36 3-5.3 攻毒………………………………………………………………………………37 第六節 Ad/M2之效力分析………………………………………………………37 3-6.1 樣本採集………………………………………………………………………37 3-6.2 母源抗體之檢測…………………………………………………………38 3-6.3 抗M2特異性抗體之檢測……………………………………………38 3-6.4 Flow cytometry……………………………………………………39 第四章結果……………………………………………………………………………………40 第一節 A/Chicken/ Taiwan /3233/04 (H5N2) 核酸序列比對…………40 第二節 M2基因選殖…………………………………………………………………40 第三節 pENTR-M2之建構………………………………………………………40 第四節 pAd-M2 plasmid之建構………………………………………40 第五節 Ad/M2之製備………………………………………………………………41 第六節 Ad/M2之M2蛋白表現…………………………………………………41 第七節 Ad/M2之免疫流程………………………………………………………42 第五章討論…………………………………………………………………………………44 第六章參考文獻…………………………………………………………………………47 表目錄 Table 1、實驗所使用之引子對……………………………………………………………………………………62 Table 2、CD4+與CD8+ T cell之flow cytometry……………………………………63 Table 3、喉頭拭子之quantitative real-time RT-PCR原始數據………63 圖目錄 Fig. 1、M2基因片段之電泳圖………………………………………………………………………64 Fig. 2、colony PCR確認pENTR-M2………………………………………………………64 Fig. 3、colony PCR確認pAd-M2 plasmid………………………………………65 Fig. 4、純化之pAd-M2 plasmid……………………………………………………………65 Fig. 5、293A細胞之CPE………………………………………………………………………………66 Fig. 6、Ad/M2之PCR確認……………………………………………………………………………67 Fig. 7、Ad/M2之病毒斑試驗…………………………………………………………………………67 Fig. 8、Ad/M2感染細胞之SDS-PAGE及西方墨點…………………………………68 Fig. 9、Ad/M2感染細胞之IFA……………………………………………………………………69 Fig. 10、Ad/M2之雞隻免疫流程…………………………………………………………………69 Fig. 11、初次免疫與補強免疫之T cell百分比……………………………………70 Fig. 12、點眼點鼻與肌肉注射之T cell百分比……………………………………71 Fig. 13、M2e ELISA結果……………………………………………………………………………71 Fig. 14、攻毒後7天死亡雞隻之病變…………………………………………………………72 Fig. 15、攻毒後7天死亡雞隻臟器之RT-PCR檢測結果.………………………73 Fig. 16、攻毒後第8天犧牲雞隻臟器之RT-PCR檢測結果……………………74 Fig. 17、喉頭拭子之RT-PCR結果………………………………………………………………75 Fig. 18、雞隻攻毒後之存活率……………………………………………………………………75
dc.language.iso	zh-TW
dc.title	腺病毒於禽流感M2疫苗發展之評估	zh_TW
dc.title	Evaluation of M2-based avian influenza virus vaccine using an adenovirus vector	en
dc.type	Thesis
dc.date.schoolyear	100-2
dc.description.degree	碩士
dc.contributor.coadvisor	鄭益謙(Ivan-Chen Cheng)
dc.contributor.oralexamcommittee	陳秋麟(Chiou-Lin Chen),闕玲玲(Ling-Ling Chueh)
dc.subject.keyword	家禽流行性感冒病毒,複製缺陷腺病毒,M2,	zh_TW
dc.subject.keyword	avian influenza virus,replication deficient adenovirus,M2,	en
dc.relation.page	75
dc.rights.note	有償授權
dc.date.accepted	2012-07-30
dc.contributor.author-college	獸醫專業學院	zh_TW
dc.contributor.author-dept	獸醫學研究所	zh_TW
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