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研究生:林沁潓
研究生(外文):Chin-HuiLin
論文名稱:腸病毒71型基因亞型間抗原決定位差異之探討
論文名稱(外文):Characterization of antigenic determinants of EV71 within intra- and inter-genotypes
指導教授:王貞仁王貞仁引用關係
指導教授(外文):Jen-Ren Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學檢驗生物技術學系碩博士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:107
中文關鍵詞:腸病毒71 型基因型抗原性微量中和試驗結合酵素免疫分析法
外文關鍵詞:enterovirus 71 (EV71)genotypeantigenicityVP1 micro-neutralization test conjugated ELISA
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腸病毒71型屬於小RNA病毒家族的一員,具有一條正股RNA基因。當被感染後,在臨床上可造成病人出現一些輕微的症狀,像是發燒、手足口症和疱疹性咽峽炎等;然而,有些感染者會引發神經性併發症,如無菌性腦膜炎、腦炎和急性弛緩麻痺,甚至會導致肺水腫及死亡。自從1969年,在加州腸病毒71型A基因型第一次被分離出來後,世界各地陸續有幾波的疫情爆發。根據腸病毒71型流行病學的研究可知,在1997年之後,基因型B3-B5以及C2-C5都曾在亞太地區造成嚴重的疫情。過去的研究已有證據指出,中和抗體在腸病毒71型感染過程中扮演保護性的角色。另外,也曾利用腸病毒71型感染的病人血清和不同基因型的病毒做中和試驗,結果發現,基因型的改變會伴隨著病毒抗原性的改變。因此,在本研究中,我們假設不同腸病毒71型基因型的抗原性改變是由於病毒基因的變化所造成的。首先,我們利用體外微量中和試驗結合酵素免疫分析法發現,臨床試驗中之疫苗病毒株免疫兔子後,其抗血清呈現出對不同腸病毒71型基因型之抗原性的差異。進一步地,我們利用健康成人以及腸病毒71型感染病患的血清,和基因型B4、B5與 C2進行試驗。結果顯示,同一血清對抗B4病毒的抗體效價較B5或C2病毒至少有高於四倍的差異,意即B4病毒的抗原性不同於B5或C2病毒。將基因型B4和B5病毒進行胺基酸序列比對發現,在病毒的外殼蛋白VP1有三個位點的變異,分別位於第98、145、164個胺基酸;其中第145和164個胺基酸變異點也同樣存在於B4和C2病毒之間。在B4和B5突變病毒抗原性實驗中,當病毒帶有VP1第98或164位點的突變時,抗體效價會有顯著的變化;而VP1 98或164伴隨145位點的雙點突變病毒,也會小幅影響抗體效價。另外,以基因重組之方式將B4和C2 VP1片段序列置換,並合成出突變病毒進行分析可發現,血清對帶C2 VP1及B4 VP1片段之突變病毒,抗體效價分別與C2及B4病毒相似,顯示VP1為主要的免疫中和區段。藉由所得的抗體效價而建構出抗原地圖可探討病毒抗原性的演化:VP1第98和164個胺基酸對於腸病毒71型的抗原特性具有重要性,且第145個位點也具有某種影響力。本研究的結果將有助於了解腸病毒71型之抗原性和疫苗的發展。
Enterovirus 71 (EV71) is a positive single-stranded RNA virus of the Picornaviridae family. It can cause mild disease such as fever, hand, foot, and mouth disease (HFMD), and herpangina, but some infections are associated with neurological complications like aseptic meningitis, encephalitis and acute flaccid paralysis. Several outbreaks were reported worldwide since it had been isolated firstly in California in 1969 known as EV71 genotype A. Studies on EV71 epidemiology showed that genotype B3-B5 and C2-C5 have caused large outbreaks in the Asia-Pacific region since 1997. Some evidences indicate that neutralizing antibody play a protective role in EV71 infection. Previous reports revealed that the genotypic shift with the changes of antigenicity by using human antisera against the various subgenotypes of EV71. In this study, we hypothesized the antigenic difference may result from the genetic variations of EV71. First, vaccine strain-immunized rabbits’ antisera were used to determine the antigenic variations among different EV71 genotypes by in vitro micro-neutralization-ELISA assay. Second, we applied healthy human and patients’ antisera against EV71 genotype B4, B5 and C2 to further investigate the viral antigenicity. The results showed that the antisera titers against genotype B4 were at least fourfold increase compared to those against genotype B5 or C2, suggesting genotype B4 had the different antigenicity from genotypes B5 or C2. Amino acid sequences comparison of genotypes B4 and B5 revealed three substitutions, position 98, 145, and 164, on capsid proteins VP1. In addition, substitutions at position 145 and 164 in VP1 were found between genotypes B4 and C2. Site-directed mutagenesis of genotype B4 or B5 virus with position 98 or 164 mutation in VP1 altered neutralizing antibody titers significantly. Besides, double mutant virus of VP1 98 or 164 along with 145 also affect the antibody titers. Antisera against mutant virus with C2 or B4 VP1 protein replacement showed neutralizing antibody titers similar to those against C2 or B4 virus, respectively, indicating that VP1 protein is the immunodominant neutralizing region. To visualize the antigenic variation of these viruses, the antigenic map was constructed. We found that the amino acid position 98 and 164 in VP1 are critical to EV71 antigenic properties and position 145 also plays a role. The results will help to understand the antigenicity of EV71 and EV71 vaccinology.
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
表目錄 VIII
圖目錄 IX
第一章、 緒論 1
一、 腸病毒之分型 1
二、 腸病毒71型之構造及複製 2
三、 腸病毒71型臨床病徵及流行病學 5
四、 腸病毒71型之檢測及診斷 8
五、 腸病毒71型之預防及治療 10
六、 腸病毒71型之免疫致病機制 11
七、 腸病毒71型之抗原結構 (antigenic structure) 12
八、 腸病毒71型中和抗體之分析平台 15
九、 病毒之感染性克隆 (infectious clone) 系統 16
十、 研究動機及目標 17
第二章、 材料與方法 19
第一節、 細胞與病毒 19
第二節、 感染性克隆 (infectious clone)之建構 21
第三節、 VP1片段置換感染性克隆之建構 28
第四節、 微量中和試驗結合酵素免疫分析法(micro-neutralization test conjugated ELISA assay) 31
第五節、 親緣演化分析 (phylogenetic analysis) 33
第六節、 抗原地圖(antigenic map)之建構 34
第七節、 統計分析 34
第三章、 結果 35
一、 比較腸病毒71型不同基因型 (genotypes) 及不同基因亞型 (sub-genotypes) 之抗原性 35
二、 血清對N7008-TW99 (B4) 和 N1745-TW08 (B5) 之中和抗體效價比較以及鞘蛋白 (capsid protein)之胺基酸序列比對 36
三、 健康成人及腸病毒71型感染病人之血清對抗N7008-TW99 (B4) 和 N1745-TW08 (B5) 與點突變病毒之中和抗體效價比較 37
四、 健康成人血清對抗N7008-TW99 (B4)及N4643-TW98 (C2)之臨床分離病毒與重組病毒中和抗體效價之比較 39
五、 基因型B和C病毒鞘蛋白(capsid protein)胺基酸序列比對以及其之間可能的抗原決定位之預測 40
六、 健康成人及腸病毒71型感染病人之血清對抗N7008-TW99 (B4) 和 N4643-TW98 (C2)與突變病毒之中和抗體效價比較 41
七、 抗原地圖(antigenic map)之建構 43
第四章、 討論 46
結語 52
參考文獻 54
表圖 63
附錄 83
自述 107


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