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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李君男(Chun-Nan Lee) | |
| dc.contributor.author | Yi-Hsing Yao | en |
| dc.contributor.author | 姚怡馨 | zh_TW |
| dc.date.accessioned | 2021-06-16T16:16:15Z | - |
| dc.date.available | 2015-03-04 | |
| dc.date.copyright | 2013-03-04 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-02-05 | |
| dc.identifier.citation | 1. Tee, K.K., et al., Evolutionary genetics of human enterovirus 71: origin, population dynamics, natural selection, and seasonal periodicity of the VP1 gene. J Virol, 2010. 84(7): p. 3339-50.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62953 | - |
| dc.description.abstract | 71型腸病毒（Enterovirus 71, EV71）屬於微小核醣核酸病毒科（Picornaviridae）中腸病毒屬（Enterovirus）的成員之一，常引起手足口症並造成小於5歲的嬰幼兒發生神經方面的併發症，嚴重者甚至會死亡。但目前並無有效疫苗上市，故研發有效的71型腸病毒疫苗極為重要。
71型腸病毒之病毒外殼由VP1、VP2、VP3及VP4蛋白質所組成，其中VP1基因序列為基因型分型之依據，可分為A、B及C基因型，而B及C基因型又可分為B1~B5 and C1~C5 等基因亞型。VP1也是病毒主要的抗原，具高度免疫性，會刺激免疫系統產生中和抗體。 腺病毒載體現在廣泛應用於表現外來基因，且有許多疫苗以此為載體。故本研究擬建構帶有VP1蛋白質基因的重組腺病毒。先前實驗室已設計五組不同組合的目標基因，即以signal peptide (SP)基因當作起始序列，中間穿插VP1基因，及用於加強免疫反應的小鼠IgG重鏈片段之基因(Fc)與霍亂毒素B次單元之基因(CT)之組合。將五種目標基因分別選殖入穿梭載體，並於大腸桿菌BJ5183中與腺病毒載體pAdEasy-1進行同質性重組。 將五種帶有目標基因之腺病毒載體分別轉染入人類胚胎腎臟細胞株（QBI-293A）中，產生重組腺病毒rADV 1/SP-VP1-Fc-CT、rADV 2/SP-VP1-Fc、rADV 3/SP-VP1-CT、rADV 4/SP- VP1及rADV 5/SP-Fc-CT。QBI-293A經重組腺病毒感染後，利用間接螢光染色來偵測腺病毒蛋白質hexon之表現，並以PCR偵測目標基因。發現五組重組腺病毒及不帶外來基因之腺病毒rADV/control皆能成功的被螢光偵測，陰性對照組則為不被病毒感染之細胞控制組。 VP1蛋白質的表現則利用間接螢光染色偵測。發現具VP1基因之重組腺病毒rADV 1/SP-VP1-Fc-CT、rADV 2/SP-VP1-Fc、rADV 3/SP-VP1-CT及rADV 4/SP-VP1都能被螢光偵測，而rADV 5/SP-Fc-CT、rADV/control及細胞控制組皆無法被偵測到螢光。 而為了偵測動物實驗之抗VP1蛋白質之血清，本研究利用大腸桿菌表現系統來大量表現VP1蛋白質，並以anti-Histidine antibody及感染過71型腸病毒之小鼠血清進行西方墨點試驗偵測，結果發現40KD位置可以偵測出與所預期之蛋白質。 接著進行動物實驗，採用口服途徑免疫週齡5~6週的BALB/c母鼠，於第1天及第15天分別給予重組腺病毒，每次劑量為100 μl，含5×108 pfu之重組腺病毒，控制組則是給予100 μl的PBS，並於第0、14、21、28天採血。接著利用ELISA試驗，將小鼠血清以1:50及1:100稀釋，以大腸桿菌表現之VP1重組蛋白質作為抗原，偵測小鼠血清中anti- VP1 IgG抗體反應。 結果顯示只有rADV 3/ SP-VP1-CT免疫的部份小鼠能產生anti- VP1 IgG,而rADV 1/ SP-VP1-Fc-CT、rADV 2/ SP-VP1-Fc及rADV 4/ SP-VP1則無此效果，而中和試驗結果顯示小鼠皆未產生能中和71型腸病毒之抗體。無法達到預期的效果，有可能是病毒劑量不足，另外亦可能是嚴苛的腸胃道環境將大部分的蛋白質之抗原決定位降解，或是重組腺病毒沒有製造出足夠量的目標蛋白質所導致。故仍需更多的研究才能確定是否可將VP1之重組腺病毒發展成疫苗。 | zh_TW |
| dc.description.abstract | Enterovirus 71 (EV71) is a human enterovirus belonging to the Enterovirus genus of the Picornaviridae family. EV71 causes hand, foot and mouth disease (HFMD) in children under 5 years old, and severe neurological complications and deaths. However, there is no effective vaccine available, so it is very important to develop vaccines against EV71.
The viral capsid is composed of VP1, VP2, VP3 and VP4. EV71 is classified into genogroups A, B, and C, and genogroups B and C are further divided into subgenogroups B1~B5 and C1~C5 based on the VP1 gene. VP1 is the main antigen of EV71 with high immunogenicity, and it may stimulate immune system to elicit specific antibody response. Adenoviral vector has been used to express target gene, and applied in vaccine development. We constructed recombinant adenoviruses containing VP1 gene. Five constructs with different combinations of target genes were generated. The target genes included the signal peptide (SP) gene, VP1 gene, mouse immunoglobulin gamma heavy chain (Fc) gene, and cholera toxin B subunit (CT) gene . Both Fc and CT were used to enhance the immune response. After the target genes were inserted into transfer vector (pShuttle-CMV), and then were transformed into BJ5183 and underwent homologous recombination with pAdEasy-1. All of these recombinant adenoviral vectors with five different targrt genes were tansfected into human embryonic kidney cell (QBI-293A cell), and recombinant adenoviruses were generated, named rADV 1/SP-VP1-Fc-CT, rADV 2/SP-VP1-Fc, rADV 3/SP-VP1-CT, rADV 4/SP- VP1, and rADV 5/SP-Fc-CT。 After QBI-293A cells were infected with recombinant adenoviruses, we used indirect immunofluorescence assay (IF) to detect the hexon protein of adenovirus and used PCR to detect the target genes. The results showed that the five different recombinant adenoviruses and rADV/control could be detected by anti-hexon protein antibody, but cell control not. The expression of VP1 protein was also deteted. It demonstrated that recombinant adenoviruses with VP1 gene (rADV 1/SP-VP1-Fc-CT, rADV 2/SP-VP1-Fc, rADV 3/SP-VP1-CT, and rADV/ 4SP-VP1) could be detected by anti-VP1 protein antibody, but rADV 5/SP-Fc-CT, rADV/control and cell control not. On the other hand, in order to detect the anti-VP1 protein antibody from mice, we used E. coli expression system to express VP1 protein. And we performed Western blot assay to detect VP1 pretein with anti-Histidine antibody and sera from EV71 infected mice. And it showed that VP1 protein could be detected with the size of 40KD. Then the animal experiment was performed. We chose 5 to 6-week-old BALB/c female mice as experimental animals, and administered two doses of recombinant adenovirus at day 1 and day 15 by oral route. Every dose included 5×108 pfu per 100 μl, and control using PBS instead. Moreover, blood was collected at day 0, 14, 21, and 28. ELISA was performed to detect the anti- VP1 IgG in mouse sera. VP1 protein expressed by E. coli system was used as antigen, and the mouse sera were diluted to 1:50 and 1:100 for testing. The results showed that only rADV 3/ SP-VP1-CT could induce a portion of the immunized mice to produce anti- VP1 IgG, but rADV 1/ SP-VP1-Fc-CT, rADV 2/ SP-VP1-Fc and rADV 4/ SP-VP1 could not. The result of neutralization test showed that no neutralizing antibodies were induced. The unpromising results may be related with not sufficient viral dosages administered. In addition, the harsh environment of digestive tract might have degraded the epitopes of VP1 protein. It is also possible that the recombinant adenoviruses did not produce enough target protein. More experiments must be done in order to determine if the VP1 recombinant adenoviruses could be vaccine candidates in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T16:16:15Z (GMT). No. of bitstreams: 1 ntu-102-R99424022-1.pdf: 5091568 bytes, checksum: d3a9d73fdb19f069749ca8d95c621a4c (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 口試委員會審定書
中文摘要--------------------------------------------------------------------------------------------i 英文摘要------------------------------------------------------------------------------------------iii 第一章 前言---------------------------------------------------------------------------------------1 1.1 71型腸病毒--------------------------------------------------------------------------------1 1.2 71型腸病毒疫苗的研發-----------------------------------------------------------------3 1.3 腺病毒載體--------------------------------------------------------------------------------4 1.3.1 腺病毒------------------------------------------------------------------------------4 1.3.2 腺病毒載體------------------------------------------------------------------------5 1.4 佐劑的應用--------------------------------------------------------------------------------6 1.5 研究目的及實驗設計--------------------------------------------------------------------7 第二章 材料與方法------------------------------------------------------------------------------8 2.1 質體的構築--------------------------------------------------------------------------------8 2.1.1 勝任細胞與質體的製備---------------------------------------------------------8 2.1.2 轉型作用---------------------------------------------------------------------------9 2.1.3挑選含外來基因之單一菌株----------------------------------------------------9 2.1.4 抽取小量質體DNA--------------------------------------------------------------9 2.1.5 次選殖基因片段-----------------------------------------------------------------10 2.1.6 限制酶酵素作用-----------------------------------------------------------------10 2.1.7 洋菜膠電泳-----------------------------------------------------------------------10 2.1.8 純化DNA-------------------------------------------------------------------------11 2.1.9 核酸連結反應--------------------------------------------------------------------11 2.1.10 核酸定序-------------------------------------------------------------------------11 2.2重組腺病毒的製備--------------------------------------------------------------------12 2.2.1 實驗系統--------------------------------------------------------------------------12 2.2.2 同質性重組反應-----------------------------------------------------------------13 2.2.3 於QBI-293A細胞中製造重組腺病毒---------------------------------------13 2.2.4 增殖重組腺病毒-----------------------------------------------------------------13 2.2.5 純化重組線病毒-----------------------------------------------------------------14 2.2.6 萃取重組腺病毒之核酸--------------------------------------------------------14 2.2.7 聚合酶連鎖反應-----------------------------------------------------------------15 2.2.8 測量重組腺病毒之力價--------------------------------------------------------15 2.2.9製造並純化大量重組腺病毒---------------------------------------------------16 2.3小鼠活體免疫試驗-----------------------------------------------------------------------17 2.3.1小鼠---------------------------------------------------------------------------------17 2.3.2免疫方式---------------------------------------------------------------------------17 2.3.3採血方式---------------------------------------------------------------------------17 2.4免疫反應分析-----------------------------------------------------------------------------18 2.4.1 酵素連結免疫吸附試驗--------------------------------------------------------18 2.4.2 中和試驗--------------------------------------------------------------------------18 2.5 蛋白質的表現與分析-------------------------------------------------------------------18 2.5.1 重組腺病毒表達目標基因-----------------------------------------------------18 2.5.2 利用大腸桿菌pLysS表現VP1 蛋白質------------------------------------18 2.5.3 表現蛋白質之分析--------------------------------------------------------------22 第三章 結果-------------------------------------------------------------------------------------25 3.1 重組腺病毒質體之構築---------------------------------------------------------------25 3.1.1 取得含目標基因之T載體----------------------------------------------------25 3.1.2 將目標基因片段選殖入穿梭載體pShuttle-CMV中---------------------25 3.1.3 產生帶有目標基因之重組腺病毒之載體----------------------------------26 3.2 製備重組腺病毒------------------------------------------------------------------------26 3.2.1 重組腺病毒之產生-------------------------------------------------------------26 3.2.2 純化重組腺病毒----------------------------------------------------------------26 3.2.3 大量製備重組腺病毒----------------------------------------------------------26 3.3 重組腺病毒於細胞株內的表現------------------------------------------------------27 3.3.1 偵測腺病毒Hexon 蛋白質---------------------------------------------------27 3.3.2 偵測71型腸病毒VP1 蛋白質-----------------------------------------------27 3.4 製備偵測抗體所用的VP1抗原蛋白質---------------------------------------------27 3.4.1 增幅目標基因--------------------------------------------------------------------27 3.4.2 將目標基因送入T載體--------------------------------------------------------27 3.4.3 將目標基因送入表現載體pRSET A-----------------------------------------28 3.4.4 誘導重組蛋白質表現-----------------------------------------------------------28 3.5 小鼠免疫反應----------------------------------------------------------------------------29 第四章 討論--------------------------------------------------------------------------------------30 圖---------------------------------------------------------------------------------------------------36 表---------------------------------------------------------------------------------------------------75 附錄------------------------------------------------------------------------------------------------77 參考文獻------------------------------------------------------------------------------------------78 | |
| dc.language.iso | zh-TW | |
| dc.subject | 重組腺病毒 | zh_TW |
| dc.subject | 71 型腸病毒 | zh_TW |
| dc.subject | 大腸桿菌表現系統 | zh_TW |
| dc.subject | VP1 蛋白質 | zh_TW |
| dc.subject | 疫苗 | zh_TW |
| dc.subject | vaccine | en |
| dc.subject | enterovirus 71 | en |
| dc.subject | E. coli expression system | en |
| dc.subject | VP1 protein | en |
| dc.subject | recombinant adenovirus | en |
| dc.title | 建構含71型腸病毒VP1蛋白質之重組腺病毒並利用大腸桿菌系統表達VP1蛋白質 | zh_TW |
| dc.title | Construction of Recombinant Adenoviruses Containing VP1 Gene of Enterovirus 71 and Expression of VP1 Protein in E. coli System | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 高全良(Chuan-Liang Kao),張淑媛(Sui-Yuan Chang) | |
| dc.subject.keyword | 71 型腸病毒,大腸桿菌表現系統,VP1 蛋白質,重組腺病毒,疫苗, | zh_TW |
| dc.subject.keyword | enterovirus 71,E. coli expression system,VP1 protein,recombinant adenovirus,vaccine, | en |
| dc.relation.page | 83 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-02-05 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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