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標題: | 利用即時反轉錄聚合酶連鎖反應分析藍舌病病毒基因體表現 Analysis of Bluetongue Virus Genome Expression by Real-Time RT-PCR |
作者: | Ting Lo 羅婷 |
指導教授: | 王汎熒(Fun-In Wang) |
關鍵字: | 藍舌病,及時反轉錄聚合酶,連鎖反應,相對定量,福馬林固定及石蠟包埋組織, Bluetongue,mRNA,Real-time RT-PCR,FFPE tissue,relative quantification, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 藍舌病是由藍舌病病毒(Bluetongue virus)所引起、以吸血昆蟲為媒介、感染反芻獸為主的疾病,其中以綿羊和白尾鹿最具感受性,但引起之臨床症狀因病毒株、動物個體及品種的影響而有差異,目前在全世界總共分離出24種血清型。 2003年台灣在金門首度分離到第二血清型藍舌病病毒,由無臨床症狀之健康山羊所分離的BTV2/KM/2003株,在Lee等人以實驗綿羊證實是一弱毒株。但國內牛及山羊的抗體陽性率偏高,分別為32.7%及8.2%,綿羊的陽性率則因地點不同變化很大,介於0 - 40%之間。
藍舌病病毒隸屬於里奧病毒科(Reoviridae)中的環狀病毒屬 (Orbivirus),無封套,雙股RNA病毒。其RNA分10個基因片段,可分別轉譯出7個結構蛋白(VP1~VP7) 與4個非結構蛋白(NS1~NS3/NS3A)。本研究的目的是由細胞內建立一個能夠定量BTV mRNA表現的方法,再應用於感染的綿羊組織(經福馬林固定及石蠟包埋)。為避免第二輪的病毒干擾,病毒感染選擇在0.1 MOI並且在感染後8小時即收成。結果顯示最高表現為NS2、 VP4 及 VP7基因的mRNA,其NS3/NS1比值為1.7。之前認為高NS3/NS1比值與病毒利用budding釋放有關。當應用於實驗感染綿羊組織,則以NS3,NS1及NS2表現最高。感染細胞與感染組織結果的不同,可能與感染時間的長短及由蠟塊萃取RNA品質不良有關。需要進一步研究才能了解mRNA表現量與BTV致病力之間的相關性。 Bluetongue is an infectious, noncontagious, arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV). BTV belongs to the Orbivirus genus of the family Reoviridae, and there are 24 recognized BTV serotypes. In Taiwan, one BTV strain of serotype 2 (BTV2/KM/2003) was first isolated from a clinically healthy goat in Kinmen in 2003, and this virus strain was defined as low virulent to experimental sheep. However the seroprevalence rates were high in ruminants, 32.7% in cattle and 8.2% in goat. That in sheep was highly variable by the locations of flocks ranged from 0 to 40%. BTV is a non-enveloped, double-stranded RNA (dsRNA) virus composed of seven structural proteins (VP1-VP7) and four non-structural proteins (NS1-NS3/NS3A). In a previous study, Huismans and Verwoerd (1973) found that the genome segments of BTV are not all transcribed at a constant rate in vitro, resulting in different amounts of BTV mRNA in infected BHK21. However the mRNAs are translated and transcribed at almost the same frequency. The purpose of this study was to establish a method of quantitating BTV mRNA expression in vitro and then applied this method to quantitate BTV mRNA expression in formalin-fixed paraffin-embedded (FFPE) tissues of experimentally infected sheep. To avoid the interference of second round virus infection, the BHK-21 cells were infected at a low MOI of 0.1 and the time point to harvest were set a 8 hr postinfection (POI). Expressions of the 10 segments of BTV genome were successfully quantitatated in vitro, wherein NS2, VP4 and VP7 mRNA were highly expressed. The expression ratio of NS3/NS1 was 1.7. Previously this large ratio is linked to a shift of virus release from lysis of cells to budding. When applied to FFPE tissues, the most highly expressed mRNA were NS3, NS1 and NS2. The different results obtained from in vitro versus in vivo models were likely due to the differences in infection duration (8 hr POI vs DPI 7), the quality of RNA from archive tissues and other factors. Further studies are needed to relate BTV mRNA expression with pathogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22346 |
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顯示於系所單位: | 獸醫學系 |
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