手足口病的『致病病毒』很多，以克沙奇病毒和腸病毒為主流。所引發的病症包括：手足水泡、口內潰瘍、以及皮疹等等。台灣30年前發生大流行，發現小於5歲幼兒被感染時，部份病童會產生重症，嚴重者甚至死亡。由於多種腸病毒引起的手足口病每年周期性的發生，基於保護幼童的立場，這是必要預防的感染症。為了對付腸病毒的肆虐，本實驗室學長鄒岳良博士研發出Ad-VLP是以腺病毒為載體的廣效手足口病疫苗。Ad-VLP疫苗可以保護基轉小鼠對抗其他手足口病病毒的致死劑量攻毒。在前輩鄒岳良博士與游舒翎博士的實驗成果顯示：Ad-VLP可以保護hSCARB2基轉小鼠免於EVA71、CVA10與CVA16的致死劑量攻毒。雖然Ad-VLP疫苗前景一片光明。但是Ad-VLP疫苗卻有一個棘手的缺點需要克服。
由於2019年COVID-19全球性大流行，AstraZeneca出產的ChAdOx1 nCoV-19疫苗和Johnson and Johnson的Janssen Ad26.COV2.S COVID-19 vaccine，把腺病毒載體式疫苗推到世人面前，也在全世界大規模的施打下，發現腺病毒載體式疫苗安全與效力表現俱佳。然而腺病毒載體式疫苗卻有個重大缺點，就是重組腺病毒製造上會產生具複製能力的腺病毒（RCA, replication-competent adenovirus），現行法規已針對RCA有明令規範。改造生產一個不會產出RAC的腺病毒載體是本論文的軸心。
我的實驗以AdEasy Adenoviral Vector System (cat. #240007)的製作模式來進行修改，利用重組位點的變換，把pAdEasy質體與修改後的pShuttle質體送入BJ5183大腸桿菌進行同源重組，產生具E1、E3、E4基因缺陷的缺陷型腺病毒質體，再送入293A進行生產病毒。期望能降低RCA的產生機率，達到US FDA的法規規範。

Hand, Foot, and Mouth Disease (HFMD) is mainly caused by Coxsackievirus and Enterovirus. Symptoms include blisters on hands and feet, mouth ulcers, and rashes. Since HFMD occurs cyclically each year, it is essential to prevent this infection, especially in young children. Currently, Dr. Zou Yueliang from our lab developed Ad-VLP, a universal HFMD vaccine using adenovirus vector. Ad-VLP can protect transgenic mice against lethal doses of other HFMD viruses. Dr. Zou Yueliang and Dr. You Shuling's results showed Ad-VLP could protect hSCARB2 transgenic mice from lethal doses of EVA71, CVA10, and CVA16.

The global COVID-19 pandemic in 2019 brought adenovirus vector vaccines like AstraZeneca's ChAdOx1 nCoV-19 and Johnson & Johnson's Janssen Ad26.COV2.S to prominence. With widespread use, these vaccines demonstrated safety and efficacy. However, a significant drawback is the production of replication-competent adenovirus (RCA). Current regulations have strict guidelines regarding RCA. The focus of this thesis is to develop an adenovirus vector that does not produce RCA.

In my experiments, I modified the AdEasy Adenoviral Vector System by altering recombination sites. The pAdEasy plasmid and modified pShuttle plasmid were introduced into BJ5183 E. coli for homologous recombination, creating a defective adenovirus plasmid with E1, E3, and E4 gene deletions. This plasmid was then used to produce the virus in 293A cells. The goal is to reduce RCA production and meet US FDA regulatory standards.

摘要 I
Abstract II
致謝 III
目錄 IV
1. 緒論 8
第一章、 腸病毒 8
1.1.1. 手足口病Hand, foot, and mouth disease （HFMD） 8
1.1.2. 引起神經受損的手足腸病毒 8
1.1.3. 腸病毒屬病毒 （Virus of the Enterovirus genus） 9
1.1.4. 腸病毒疫苗的發展 10
第二章、 腺病毒載體 11
1.1.5. 腺病毒（adenovirus）的發現與疫苗發展史 11
1.1.6. 重組腺病毒（Recombinant adenoviruses, rAds）的應用及發展 11
1.1.7. 重組缺陷型腺病毒的製造 13
1.1.8. Replication-competent adenovirus （RCA） 14
1.1.9. 腺病毒的構型 15
1.1.10. 腺病毒的複製週期 15
1.1.11. 腺病毒的基因 16
1.1.12. 早期轉錄基因（Viral early unit, E-） 17
1.1.13. 後期轉錄基因（Viral late unit, L-） 19
2. 研究動機與設計 19
研究動機 19
研究設計 20
研究步驟 20
3. 實驗方法與材料 21
3.1. 生產病毒 21
3.2. 動物實驗 21
3.2.1. 疫苗施打 21
3.2.2. 取得血清 21
3.2.3. CVA6攻毒 22
3.3. 質體建構 22
3.3.1. pShuttle-CMV-eGFP質體（附件六） 22
3.3.2. pShuttle-pL5質體（附件七） 22
3.3.3. pShuttle-pL5-S-D質體（附件八） 22
3.3.4. pShuttle-FL5質體（附件九） 23
3.3.5. pShuttle_FL5_S_D質體（附件十） 23
3.3.6. pShuttle-FL5-eGFP質體（附件十一） 23
3.3.7. pShuttle_FL5_FL5質體（附件十二） 24
3.3.8. pAd-dE4-S-D質體（附件十三） 24
3.3.9. pAd-dE4-eGFP質體（附件十五） 24
3.3.10. pAd-eGFP質體（附件十六） 24
3.3.11. pcDNA_E4質體（附件十七） 24
3.3.12. pSBtet_E4質體（附件十八） 25
3.3.13. pSBtet_E4_CMV_L5質體（附件十九） 25
3.3.14. Polymerase chain reaction （PCR）-使用ALLin Mega HS HiFi DNA Polymerase （cat. HLE0401） 26
3.3.15. Polymerase chain reaction （PCR）-使用Phusion High-Fidelity DNA Polymerase （cat. F530S） 27
3.3.16. Colony-PCR-使用ALLin Mega HS HiFi DNA Polymerase （cat. HLE0401） 28
3.3.17. 質體抽取-使用FavorPrep Plasmid Extraction Mini Kit （cat. FAPDE100） 29
3.3.18. 膠體純化-使用FavorPrep Gel/PCR Purification Mini Kit （cat. FAGCK001） 29
3.3.19. PCR純化-使用FavorPrep Gel/PCR Purification Mini Kit （cat. FAGCK001） 30
3.3.20. Optimizing Restriction Endonuclease Reactions 30
3.3.21. Ligation 30
3.3.22. Transformation 30
3.3.23. Electroporation Transformation 31
3.3.24. Establish Chemically Competent Cell - ChampiomTM E.coli Transformation Kit（Cat. CK1000） 31
3.3.25. Establish Electroporation Transformation BJ5183（Cat. 200154）Competent Cell 31
3.3.26. 凍菌保存 32
3.4. 抽取mRNA - RNAzol® RT （Cat. RN 190） 32
3.5. 抽取Protein 32
3.6. Reverse Transcription cDNA - SuperScript™ III Reverse Transcriptase （Cat. No.18080-093） 33
3.7. Real-Time PCR - KAPA SYBR® FAST qPCR Kit Optimized for LightCycler® 480 （cat. KK4609） 35
3.8. Transfection - Lipofectamine™ 3000 （cat. L3000015） 36
3.9. Transfection - Thermo Scientific TurboFect Transfection Reagent （cat. R0531） 37
3.10. Establish Packaging Cell Line 293A 37
3.11. Western Blot 38
3.12. ELISA 39
3.13. BCA Assay 39
4. 實驗結果 40
4.1. CVA6的病毒生產 40
4.2. Ad-VLP的抗體結合CVA6的能力 40
4.3. Package Cell Line的蛋白質表達 40
pcDNA-E4 293A： 40
pSBtet-E4 293A： 41
pSBtet-E4-CMV-L5 293A： 41
4.4. 質體建構和腺病毒的生產 41
pshuttle-pL5和pshuttle-pL5-S-D 42
pshuttle-FL5和pshuttle-FL5-S-D 42
pshuttle-FL5-eGFP 42
pshuttle-FL5-FL5 43
pAd-dE4-eGFP + pshuttle-FL5-FL5 43
pSBtet-E4-CMV-L5 293A 43
購買新的pAdEasy-2（V005925）（附件四）實驗組與pAdEasy-1(#240005)（附件二）控制組 44
pAdEasy-1控制組 44
新的pAdEasy-2實驗組 45
5. 討論 45
5.1. Ad-VLP的抗體結合CVA6的能力 45
5.2. Package Cell Line的基因表達測定 46
5.3. 質體設計 46
6. 圖表 47
7. 附件 91
8. 參考文獻 111

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