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系統識別號 U0026-0812200912011804
論文名稱(中文) 3D聚合酶突變對腸病毒71型複製所造成的影響
論文名稱(英文) The effect of 3D polymerase mutation on enterovirus 71 replication
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 龔彥樺
研究生(英文) Yen-Hua Kung
學號 t3693401
學位類別 碩士
語文別 中文
論文頁數 82頁
口試委員 口試委員-劉校生
口試委員-王憲威
指導教授-王貞仁
中文關鍵字 腸病毒71型  3D聚合酶 
英文關鍵字 3D polymerase  enterovirus 71 
學科別分類
中文摘要   腸病毒71型(Enterovirus 71)1998年於台灣爆發大流行,出現 78名死亡個案。目前對於腸病毒71型的致病機轉、毒力決定因子尚有許多不了解的地方。在小兒麻痺病毒的研究中,已證實轉譯出RNA聚合酶的3D區域突變會造成小兒麻痺病毒複製能力及毒力的改變。因此本研究主要探討3D區域胺基酸的突變對腸病毒71型複製能力之影響。
  腸病毒71型之臨床病毒株在1998年大流行前後造成的臨床症狀嚴重度有極大的差異,比較其3D區域序列後發現,在第251個胺基酸位置,1998年多屬纈胺酸(valine,V)及異白胺酸(isoleucine,I),但1986年則為酥胺酸(threonine,T)。另外,根據文獻指出,小兒麻痺病毒3D區域328的天門冬胺酸(aspartic acid,D)突變後會嚴重影響小兒麻痺病毒3D聚合酶的功能。上述的胺基酸差異是否造成腸病毒71型複製上的改變,進一步利用定點突變的方式,在複製子(replicon)及感染性克隆(infectious clone)的3D區域進行I251T及D328H的突變來探討。實驗結果發現,D328H突變的複製子,其所表現的螢火蟲螢光素酶活性(firefly luciferase activities)明顯降低許多,顯示328胺基酸位置在腸病毒71型3D聚合酶功能上扮演重要的角色。但D328H的感染性克隆能在轉染後第八天偵測到病毒顆粒產生,出現突變回復(reversion)的現象。從此現象我們推測D328H突變之腸病毒71型病毒株還具有微弱的複製能力,持續複製到突變回復發生。
  而I251T突變的複製子或感染性克隆在35℃下,其複製能力和野生型無太大差異。但在39.5℃高溫下其複製能力明顯較野生型緩慢。此研究結果顯示1986年臨床病毒株其3D區域的251位置為T時,在高溫下複製能力較差,呈現嚴重的溫度敏感性(strong temperature sensitive)現象,可能較不易造成嚴重的神經症狀。總之,本研究證實3D區域的突變會影響腸病毒71型的複製能力:328胺基酸位置突變會嚴重影響腸病毒71型的複製,而251胺基酸位置由I突變為T時則會造成病毒在高溫下複製能力減低。




英文摘要 Enterovirus 71 (EV71) epidemic resulted in 78 deaths in 1998 in Taiwan, with an average of 40 fatalities each year, but the molecular basis of EV71 pathogenicity remains poorly understood. Studies in poliovirus have suggested mutations in 3D polymerase region resulted in changing growth rate, viral RNA accumulation, temperature susceptibility, and attenuation in mice. Therefore, we hypothesized that 3D region may be one of the major factor altering biological characteristics and virulence of EV71. The aims of this study are to examine the effect of mutations on biological properties of 3D by site-direct mutagenesis using EV71 replicon and infectious clone systems. Two strategies were used to examine whether mutations in 3D region have the potential to affect 3D polymerase function. First, sequences of clinical isolates from 1986 and those from or after 1998 outbreaks were compared. Amino acid sequence of 3D of isolates from or after 1998 epidemic showed T251V or T251I mutation when compared with 1986 isolates. Secondly, those well-defined mutations with measurable phenotypic defects in poliovirus were examined in EV71 3D region. It was known that aspartic acid (D) in position 328, located in YGDD motif, is important for 3D polymerase activities of poliovirus. In this study, two mutations, D328H and I251T were introduced into EV71 replicon and infectious clone. The luciferase activity and virus replication rate of D328H are dramatically decreased when compared with wild type. However, this D328H mutant infectious clone was found to have a reversion from 3D-H-328 to 3D-D-328 (wild-type) at day 8 post transfection. In the I251T mutant, luciferase activities and viral replication rate showed no significant difference at 35℃ when compared with wild type, but I251T mutant showed lower luciferase activities and viral replication rate than wild type when they were incubated at 39.5℃. The results suggested that D328H mutant 3D polymerase can support a low level replication to generate a reversion, and thronine at position 251 results in strong temperature sensitivity may contribute to attenuation in virulence of clinical isolates.



論文目次 中文摘要…………………………………………………………… Ⅰ
英文摘要…………………………………………………………… Ⅲ
誌謝………………………………………………………………… Ⅴ
目錄………………………………………………………………… Ⅵ
表目錄……………………………………………………………… Ⅷ
圖目錄……………………………………………………………… Ⅷ
第一章、緒論
一、腸病毒71型的分類及構造………………………………………… 1
二、腸病毒的臨床表徵及流行歷史…………………………………… 2
三、腸病毒的毒力研究………………………………………………… 7
四、腸病毒的轉譯、複製……………………………………………… 8
五、病毒的RNA-dependent RNA polymerase—3D聚合酶…………… 10
六、感染性克隆(infectious clone)與複製子(replicon)系統………14
七、研究目標…………………………………………………………… 16
第二章、材料與方法
一、細胞與病毒………………………………………………………… 17
二、腸病毒71型3D區域之比對………………………………………… 20
三、質體DNA的操作…………………………………………………… 22
四、RNA之製備及轉染………………………………………………… 24
第三章、結果
一、腸病毒71型臨床分離株之生物特性分析………………………… 27
1. 溫度感受性分析……………………………………………………… 27
2. 生長速率分析………………………………………………………… 28
3. 腸病毒71型溫度敏感性及溫度耐受性病毒株3D區域的定序及比對 29
4. 1998年大流行前後之腸病毒71型病毒株3D區域的比對…………… 30
二、小兒麻痺病毒3D區域的探討……………………………………… 31
三、複製子及病毒轉殖感染株突變對複製的影響…………………… 32
1. 突變複製子及病毒轉殖感染株的建構……………………………… 32
2. 突變複製子及病毒轉殖感染株的RNA之製備……………………… 33
3. 突變複製子RNA轉染於SK-N-SH細胞………………………………… 34
4. 突變病毒轉殖感染株RNA轉染於SK-N-SH細胞……………………… 36
5. 突變病毒之生物特性分析…………………………………………… 38
6. D328H突變病毒之突變回復………………………………………… 38
第四章、討論………………………………………………………………40
參考文獻………………………………………………………………… 49
圖表…………………………………………………………………………54
附錄…………………………………………………………………………78
自述…………………………………………………………………………82
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