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研究生:許廣治
研究生(外文):Kuang-Chih Hsu
論文名稱:埃及斑蚊感染登革病毒第1型與第4型後免疫基因的變化及病毒量的改變
論文名稱(外文):Viral titers and mosquito immune gene changes post Aedes aegypti infected with Dengue virus serotype 1 and 4
指導教授:陳建先陳建先引用關係
指導教授(外文):Chien-Hsien Chen
口試委員:陳建先
口試委員(外文):Chien-Hsien Chen
口試日期:2015-07-28
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
論文頁數:59
中文關鍵詞:DVRF2螢光靶即時定量聚合酶鏈鎖反應defensin A1defensin A4cecropin B1DVRF1Vago
外文關鍵詞:DVRF2one-step probe real time RT-PCRdefensin A1defensin A4cecropin B1DVRF1Vago
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登革熱和登革出血熱,是台灣以及世界上許多地方的主要公共衛生問題。登革熱在台灣的流行,主要是藉由在南台灣分布的埃及斑蚊傳播,次要則為全台都有的白線斑蚊傳播。病媒蚊埃及斑蚊與白線斑蚊兩種蚊子在感染登革病毒後,每天病毒效價的變化,也是本研究的課題。本實驗以螢光靶即時定量聚合酶鏈鎖反應,可以專一性、敏感性與定量性偵測到登革病毒第1型與第4型,並以實驗室飼養的埃及斑蚊與白線斑蚊雌成蚊經胸側接種登革病毒第1型與第4型後做檢測,結果登革熱病毒量隨天數增加而增加,第8天或第10天達到最高量(登革第1型病毒量8.1X105,登革第4型病毒量 2.8X106),隨後遞減,登革病毒NS1蛋白也是同樣的表現模式,第8天或第10天達到最高量。
在埃及斑蚊感染登革病毒後,相關的免疫基因之變化,也是本實驗研究的重點。其中defensin A1, defensin A4, cecropin B1是蚊子等昆蟲類的原始免疫基因,可以用來對抗外來的微生物,結果顯示此3種基因,除第1天外均呈現增加趨勢。另外蚊體之DVRF1, DVRF2基因,文獻(Souza-Neto,J.A.2009)報導可以對抗登革病毒,本研究顯示蚊子感染後DVRF2基因是增加的,但DVRF1是被抑制。文獻(Paradkara , et al.2012)指出Vago基因在家蚊類可以對抗西尼羅河病毒,但本研究顯示登革病毒感染後是受抑制的。總結:埃及斑蚊感染登革病毒後原始免疫基因是增加的,但DVRF1,Vago基因受抑制。
Dengue fever and Dengue hemorrhagic fever are major public health burden in Taiwan and many parts of the world. These diseases are transmitted mainly by Aedes aegypti mosquito in southern part and secondary by Aedes albopictus in whole Taiwan Island. The viral loads of mosquito each day after Aedes aegypti and Aedes albopictus mosquitoes infected with dengue viruses were the topic aim. In this study, we use one-step probe real time RT-PCR method to detect dengue virus serotype 1 and serotype 4 intra-thoracical infected mosquitoes, and the results revealed that virus titers were increased with days and highest on day 8 or 10(DV1 Viral titers 8.1X105,DV4 Viral titers 2.8X106). The Western blot also revealed same pattern that dengue NS1 protein is more prominent on day 8 or 10.
The changes of immune related genes expression in mosquito post dengue virus infection were also our research aim. Of these mosquito innate immune genes, defensin A1, defensin A4 and cecropin B1 can be expressed to against microorganism, such as Gram negative and positive bacteria. Our results revealed these 3 genes were all increased post dengue virus infection, except on day 1. The DVRF1 and DVRF2 gene were reported (Paradkara , et al.2012) for mosquito to confront dengue virus infection; our results revealed that DVRF1 is inhibited, while DVRF2 is induced post dengue virus infection. Vago gene in Culex mosquito is reported (Souza-Neto,J.A.2009) to antagonize West Nile virus, but our result was that it was inhibited in Aedes aegypti mosquito post dengue virus infection. All of above study showed the genes expressions of mosquito innate immune genes ( defensin A1, defensin A4, cecropin B1, and DVRF2 ) were increased post virus infection, while DVRF1 and Vago genes were inhibited post dengue virus infection.
Keywords:one-step probe real time RT-PCR, defensin A1, defensin A4, cecropin B1, DVRF1, DVRF2, Vago
誌謝 i
摘要 iii
ABSTRACT iv
目次 vi
表次 ix
圖次 x
附錄 xi
第壹章 緒論 1
第一節 研究動機 1
第二節 研究目的與問題 2
第貳章 文獻探討 4
第一節 登革熱病毒 4
第二節 蚊子的先天性免疫反應 6
第三節 TOLL/DIF , IMD/REL , JAK STAT PATHWAYS 相關機制簡介 8
第參章 研究設計 10
第一節 蚊子飼養 10
第二節 實驗細胞培養 10
第三節 病毒培養與病毒溶斑試驗(PLAQUE ASSAY) 10
一、 病毒的培養(VIRUS AMPLIFY) 10
二、 病毒溶斑試驗(PLAQUE ASSAY) 11
第四節 登革熱病毒接種於蚊子 12
第五節 蚊子的RNA分離與反轉錄CDNA 12
第六節 登革熱病毒檢測設計專一性引子和螢光靶 12
第七節 以螢光靶即時定量聚合酶鏈鎖反應檢測病毒RNA效價 13
第八節 標準CRNA(登革熱基因以聚合酶鏈鎖反應置入PGEMT-EASY載體)製備 13
第九節 設計檢測埃及斑蚊基因之專一性引子 14
第十節 蚊子基因之專一抗體製備 14
第十一節 蛋白質定量 (DETECTION OF PROTEIN CONCENTRATION) 14
第十二節 西方墨點轉漬法 (WESTERN BLOT) 15
一、 蛋白質電泳 15
二、 轉漬(TRANSFER) 15
三、 ECL DETECTION 15
第肆章 研究結果 17
第一節 登革病毒測試 17
第二節 登革病毒於蚊體內量變化 17
第三節 登革病毒於蚊體內免疫基因變化 17
第四節 登革病毒與蚊體內免疫基因蛋白表現 18
第伍章 討論與建議 19
第一節 討論 19
第二節 後續研究建議 20
第三節 結論 20
參考文獻 21
中文部份 21
英文部份 22
附表 25
附圖 31
附錄 38
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