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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 高全良(Chuan-Liang Kao) | |
| dc.contributor.author | Fai Hon | en |
| dc.contributor.author | 韓暉 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:52:58Z | - |
| dc.date.available | 2021-12-31 | |
| dc.date.copyright | 2011-03-03 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-02-12 | |
| dc.identifier.citation | 1. Murphy, E., et al., Reevaluation of human cytomegalovirus coding potential. Proceedings of the National Academy of Sciences of the United States of America, 2003. 100(23): p. 13585-13590.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48335 | - |
| dc.description.abstract | 人類巨細胞病毒（Human cytomegalovirus, HCMV）屬於皰疹病毒科（Herpesviridae）。一般健康成人感染巨細胞病毒並不會產生臨床症狀，但對免疫系統受損的病人或免疫功能尚未發展完全的嬰兒，巨細胞病毒會造成肺炎、肝炎、腦炎等嚴重疾病，並造成很高的致死率。
目前治療巨細胞病毒可以ganciclovir等藥物抑制病毒複製，但有研究指出對ganciclovir等藥物具抗藥性的病毒株已經產生。Intravenous Immunoglobulin (IVIG)近年被發現在臨床應用上可有效地預防胎兒受到感染。IVIG是從超過一千人的混合血漿中純化出來的免疫球蛋白（IgG），有中和體內HCMV病毒的效用。IVIG的製備必須先大量篩選含有HCMV中和抗體的血清，但中和試驗的操作過程複雜且十分費時，並不適合用於大量篩選，因此有研發新的中和抗體測試方法的必要性。我們希望可以藉由表現HCMV重組蛋白質建立酵素連結免疫吸附試驗(ELISA)取代傳統的中和試驗。 在人體感染HCMV後產生中和抗體的研究，最廣為報導會產生中和抗體的特異性抗原是B醣蛋白質 (glycoprotein B)，而其中AD1（antigenic domain 1）和AD2（antigenic domain 2）更是大部分中和抗體所結合的位置。為了解臺灣HCMV臨床株在此兩個區域的基因差異性，我們從臺大醫院病毒室取得病毒臨床株進行gB AD1和AD2的序列分析。結果顯示AD1的變異性較低(相似度98.58%)，而AD2的變異性較大(相似度87.83%)，與前人研究相符。種系分析則顯示臺灣臨床株的AD2可劃分為兩個族群，而歐洲國家則有與臺灣臨床株差異較大的族群。由於上述兩個domain是產生中和抗體的重要抗原，且AD2的基因變異可分成兩個族群，因此製造AD1和包含二株不同病毒的AD2重組蛋白質預期可以達到偵測中和抗體的效果。本研究選用baculovirus表現系統表現目標蛋白質，希望得到的重組蛋白質可以被醣化而增加抗體偵測的敏感度。 本研究使用臺大醫院分離出來的HCMV病毒株TW71與參考病毒株AD169作為基因的來源，先用PCR方法分別增幅AD169 gB signal peptide (Sig)、gB AD1、gB AD2和TW71 gB AD2基因片段，然後把基因分別連接成AD169 gB Sig-AD1和gB Sig-AD2AD169-TW71並建構在轉移載體上，確認序列沒有錯誤後利用桿狀病毒蛋白質表現系統製造基因重組桿狀病毒，並感染昆蟲細胞得到重組蛋白質。為了瞭解蛋白質被醣化後是否可以提高抗體偵測的敏感度，我們同時構築了不帶有signal peptide的AD2AD169-TW71，使其製造出來的蛋白質不被醣化。利用人類血清抗體進行西方點墨法，結果顯示此三種重組蛋白質均具有抗原性。使用純化之Sig-AD2重組蛋白質建立ELISA偵測HCMV血清抗體陽性和陰性檢體，得到陽性率為64.29 %，專一性為100 %。將ELISA的數據與中和試驗的結果進行相關性分析後得到相關系數為0.4477，而與商用EIA結果比較的相關系數則為0.7407，Sig-AD2 ELISA方法所檢測的數值普遍比EIA的結果低。綜合以上實驗結果，顯示單獨的Sig-AD2抗原所建立之偵測方法的敏感度並不足以用作中和抗體的偵測，未來期望加入Sig-AD1抗原共同偵測可以提高ELISA的敏感度，且預期可以比商用EIA的方法更能接近中和試驗的結果。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:52:58Z (GMT). No. of bitstreams: 1 ntu-100-R97424004-1.pdf: 2720073 bytes, checksum: 649d1fd50191ad9c7f040492227e4814 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員會審定書.....................................................i
致謝.................................................................ii 中文摘要.............................................................iii 英文摘要.............................................................v 第一章 緒論.........................................................1 第一節 人類巨細胞病毒 一、病毒構造.............................................1 二、病毒的基本特性.......................................1 三、病毒的複製...........................................2 四、致病機制.............................................3 五、血清流行病學.........................................4 六、抗人類巨細胞病毒的藥物治療和預防.....................4 七、免疫球蛋白製劑.......................................5 八、人類巨細胞病毒醣蛋白質B.............................6 第二節 桿狀病毒 一、病毒結構.............................................7 二、病毒的複製...........................................7 三、利用桿狀病毒作為蛋白質表現系統.......................9 第三節 研究目的................................................10 第二章 材料與方法..................................................12 第一節 實驗材料................................................12 一、試劑、試藥..........................................12 二、商用套組............................................13 三、細胞株..............................................14 四、病毒株..............................................14 五、血清檢體............................................15 第二節 實驗方法.................................................15 一、試劑之配製..........................................15 二、細胞培養............................................21 三、病毒培養............................................23 四、偵測病毒力價........................................24 五、病毒核酸之抽取......................................26 六、巨細胞病毒醣蛋白質B基因之構築......................26 七、製造攜帶目標基因之重組桿狀病毒......................30 八、利用重組病毒進行蛋白質表現..........................33 九、蛋白質偵測..........................................34 十、蛋白質純化..........................................36 十一、ELISA酵素連結免疫吸附試驗........................37 十二、中和試驗..........................................37 第三章 實驗結果....................................................39 第一節 酵素免疫法與中和試驗的相關性比較.........................39 第二節 人類巨細胞病毒醣蛋白質B AD1、AD2核酸序列分析..........39 第三節 重組基因的建構...........................................40 第四節 製造重組桿狀病毒.........................................40 第五節 重組桿狀病毒感染細胞之能力...............................41 一、Bac-Sig-AD1和Bac-Sig-AD2AD169-TW71...................41 二、Bac-AD2AD169-TW71-12G1...............................41 第六節 重組蛋白質的特性預測.....................................41 第七節 表現重組蛋白質...........................................42 一、重組蛋白質的表現位置偵測.............................42 二、使用人類血清偵測重組蛋白質...........................43 三、比較不同細胞株表現重組蛋白質的效果...................44 四、重組蛋白質的最適化表現條件...........................44 五、重組蛋白質的純化......................................44 六、重組蛋白質的產量.....................................45 第八節 酵素免疫法的建立.........................................45 一、Goat anti-human IgG conjugated AP的最適合稀釋濃度.......45 二、測試重組蛋白質之最適合coating濃度....................46 三、以人類血清進行酵素免疫法測試蛋白質的抗原性...........46 四、使用Sig-AD2AD169-TW71重組蛋白質建立酵素免疫法.........47 五、以Sig-AD2AD169-TW71抗原建立之ELISA與血清中和效價之 相關性比較...........................................47 第四章 討論........................................................48 第一節 gB AD2序列之變異性與血清中和效價之關係.................48 第二節 影響桿狀病毒載體的蛋白質表現能力之因素..................49 第三節 重組蛋白質的特性........................................49 第四節 重組蛋白質之表現與純化..................................51 第五節 重組蛋白質於抗體檢測上之應用............................52 參考文獻............................................................94 附錄...............................................................102 | |
| dc.language.iso | zh-TW | |
| dc.subject | 蛋白表現 | zh_TW |
| dc.subject | 巨細胞病毒 | zh_TW |
| dc.subject | 醣蛋白B | zh_TW |
| dc.subject | 中和抗體 | zh_TW |
| dc.subject | 中和試驗 | zh_TW |
| dc.subject | 酵素免疫吸附試驗 | zh_TW |
| dc.subject | 桿狀病毒 | zh_TW |
| dc.subject | glycoprotein B | en |
| dc.subject | protein expression | en |
| dc.subject | baculovirus | en |
| dc.subject | ELISA | en |
| dc.subject | neutralization test | en |
| dc.subject | neutralizing antibody | en |
| dc.subject | CMV | en |
| dc.title | 以桿狀病毒表現載體於昆蟲細胞表現人類巨細胞病毒gB AD1及AD2蛋白質片段及其在抗體檢測上之應用 | zh_TW |
| dc.title | Expression of Human Cytomegalovirus Glycoprotein B AD1 and AD2 Fragments in Insect Cells by Baculovirus Expression System and Its Application in Antibody Detection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李君男(Chun-Nan Lee),張淑媛(Sui-Yuan Chang),吳宗遠 | |
| dc.subject.keyword | 巨細胞病毒,醣蛋白B,中和抗體,中和試驗,酵素免疫吸附試驗,桿狀病毒,蛋白表現, | zh_TW |
| dc.subject.keyword | CMV,glycoprotein B,neutralizing antibody,neutralization test,ELISA,baculovirus,protein expression, | en |
| dc.relation.page | 106 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-02-14 | |
| 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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