Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52700
標題: 家禽里奧病毒毒力因子探討
Investigation on the Virulence Factor of Avian Reovirus
作者: 李龍湖
王敏盈
關鍵字: 基礎研究
Avian reovirus
畜牧獸醫類
家禽里奧病毒
雙股RNA
反向遺傳學系統
dsRNA
reverse genetics system
摘要: 本計劃擬利用反向遺傳學系統 (reverse genetics system ) 技術探討家禽里奧病毒 (ARV) 腳底接種後,S1133毒株引起關節炎或2408毒株引起急性死亡之毒力因子。擬分三年進行。第一年著重於ARV S1133 及2408 各10段RNA基因體片段之全長cDNA製造並嵌入pcDNA 3.1 plasmid中,構築上游含T7 啟動子序列及下游含D 型肝炎病毒終止密碼序列之pT7-ARVcDNA-HDVR 之重組質體,其中10 源自S1133 毒株;另10 種則源自2408毒株。另外構築T7聚合酶重組質體pT7-pol. 於重組質體鑑定後,利用共同轉染( cotransfection )及基因 reassortment 原理,製備包括9 段源自S1133毒株RNA 及1 段源自2408 毒株之重組病毒10 株;以相同方法製備包括9段源自2408 毒株RNA 和1 段源自S1133 毒株之重組ARV 10 株。接著以SPF 雞接種分析試驗,鑑定ARV 哪一段RNA 基因體交換後,會導致病原顯著改變,以確定哪一段基因體 (簡稱 X片段)和病徵有關。接著構築分別來自S1133及2408毒株X片段之chimeric X cDNA片段。並依上述相同方法構築成重組質體pT7-chimeric X cDNA-HDVR 。再和pT7-ARVcDNA-HDVR以外的9種重組其體及pT7-pol共同轉染以製造chimeric重組ARV。再接種SPF雞隻,分析chimeric 重組ARV 病原性的改變,從而得知ARV X片段RNA哪些區域和ARV導致病徵相同。最後再利用點變異技術,將上述和毒力因子相關區域內的毒株間差異的胺基酸進行點變異,進而以相同方法製造出點變異病毒株,再接種SPF雞隻分析ARV X片段RNA 哪些胺基酸和病毒毒力相關,從而鑑定ARV 致病毒力因子。
Avian reovirus (ARV) S1133 has been reported as an etiological agent. Recently, Wefound that ARV 2408 could cause high mortality in infected birds with coagulativenecrosis lesions in liver, at 2-3 days postinoculation. Thus, this research proposal isto analyze the virulence factor involved in arthritis or acute death caused by ARV,using a reverse genetics system. The time table for this proposal includes 3years. Inthe first year, cDNA of 10 segments of each of ARV S1133 or 2408 genomic RNAwill be synthesized and cloned into pcDNA3.1 plasmid to generate a series ofrecombinant plasmids (RP) (pT7-ARV cDNA-HDVR), which includes T7 promoterupstream of ARV cDNA and hepatitis D virus ribozyme ( HDRV ) sequence. A totalof 20 RP will be constructed, 10 RP from each of 10 genome segments of S1133 and10 RP from each of 10 genome segments of 2408. In addition, a RP (pT7-pol) whichincludes T7 polymerase will also be constructed in this study. Nine RPs of ARVS1133, one RP of 2408, and T7-pol will be used to cotransfect into DF-1 cell line togenerate 10 recombinant viruses (RV). The genomic RNA segments of each theseRV includes 9 RNA from S1133 and one segment RNA from 2408. Following thesame procedures, 10 RV will be generate. The genome of each these RV include 9RNA segments from 2408 and one RNA from S1133. These RV will be used toinfect SPF birds to determine which viral RNA segment ( called X RNA segment )involves in virulence. In the second year, to further determine if region(s) of X RNAsegment involve(s) in virulence factor(S), chimeric X cDNA fragments derived fromS1133 and 2408 X RNA segment will be constructed to generate chimeric RP(pT7-chimeric X cDNA-HDVR). Nine RPs of S1133, one RP of chimeric X cDNA,and pT7-pol will be used to cotransfect cells to generate chimeric RV. The sameprocedures will be used to generate chimeric RV using 9 RP of 2408 forcotransfection. The region of X RNA segment which involves virus virulent factorwill be assessed in birds infected with the chimeric RV. Finally, to generated pointmutation RV to determine if amino acid(s) in the region of X RNA segmentinvolve(s) in virulence factor of ARV, point mutation of amino acid in the region ofX RNA segment will be changed to generate point mutant RV. These RV will beused to inoculate birds for the evaluation of ARV virulence. The amino acid-basedvirulence factors will be finally determined.
URI: http://hdl.handle.net/11455/52700
其他識別: NSC97-2313-B005-005-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1664934&plan_no=NSC97-2313-B005-005-MY3&plan_year=97&projkey=PD9709-0235&target=plan&highStr=*&check=0&pnchDesc=%E5%AE%B6%E7%A6%BD%E9%87%8C%E5%A5%A7%E7%97%85%E6%AF%92%E6%AF%92%E5%8A%9B%E5%9B%A0%E5%AD%90%E6%8E%A2%E8%A8%8E
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