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  2. 獸醫學院
  3. 微生物暨公共衛生學研究所
Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66351
標題: 反向遺傳技術伴隨野外強毒株感染加速傳染性華氏囊病毒之細胞馴化
Pathogenic bursa-derived field strain contributes to rescue cell-culture-adapted infectious bursal disease virus using RNA polymerase II reverse genetics strategy
作者: 陳政延
Chen, Jeng-Yan
關鍵字: infectious bursal disease virus;傳染性華氏囊病毒;reverse genetics;反向遺傳技術
出版社: 微生物暨公共衛生學研究所
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摘要: 
傳染性華氏囊病 (infectious bursal disease；IBD) 由傳染性華氏囊病毒 (IBDV) 所引起，在幼齡雞群中是一種高度傳染性的疾病。IBDV 屬於不具封套的病毒顆粒，基因是由雙股螺旋的片段 A 和片段 B 所組成，其長度分別為 3.3 kb 和 2.9 kb。由於從雞身上分離出的超強毒株 (very virulent IBDV；vvIBDV) 無法輕易的馴化至細胞株，故需將此病毒長時間在細胞株持續繼代，才有可能在細胞馴化成功，但並非對每一分離株皆可得到預期的結果。文獻指出將 IBDV 的外部殼蛋白 VP2 上的兩個特殊胺基酸進行突變後，可使 IBDV 馴化至細胞株，此兩特殊胺基酸分別為第 253位置的穀胺醯胺 (glutamine)，和第 284 位置的丙胺酸 (alanine)。因此本實驗目的是以 vvIBDV-TWN 基因為基礎利用 RNA 聚合酶 II 之反向遺傳技術，期望能產生出細胞馴化之 vvIBDV。先前的實驗嘗試將 vvIBDV-TWN 病毒株馴化至細胞株，結果顯示即使經過多次之盲目繼代，此野外株無法感染細胞。因此本實驗分別建構出具有 vvIBDV-TWN 病毒株的片段 A 和片段 B及 hepatitis delta virus ribozyme (HDR) 之 pVAX/SegA/HDR 和 pVAX/SegB/HDR 重組質體。另外也建構出 pVAX/mutSegA/HDR 重組質體，此質體具有在 VP2 上第 253 和 284 位置產生的 Q253H 和 A284T 兩位置之突變。本實驗將不同組合的重組質體轉染至 DF-I 細胞，另也嘗試質體轉染後，進行 vvIBDV-TWN 病毒株之感染。結果顯示只要包含 pVAX/mutSegA/HDR 質體的轉染組合，並伴隨 vvIBDV-TWN 病毒株之感染，皆能發現細胞病變效應。經由間接免疫螢光分析，病變之細胞呈現出陽性的螢光訊號。收集含有細胞病變的細胞上清液，以 reverse transcription- polymerase chain reaction 作用後，再經由重組病毒上才具有的 BglII 切位的酵素切割，即可確認增幅產物是源自 pVAX/mutSegA/HDR 轉染的重組病毒。實驗結果指出藉由 RNA聚合酶 II 的反向遺傳技術，在 vvIBDV-TWN 病毒株感染下，能產生出細胞馴化之 IBDV。

Infectious bursal disease virus (IBDV) causing a highly contagious disease in young chickens is composed of non-enveloped capsid containing two segments of double stranded RNA, Segment A (3.3 kb) and Segment B (2.9 kb). Pathogenic wild-type very virulent IBDV (vvIBDV) was not easy to be adapted to cells. In previous studies, the vvIBDV-TWN strain could not be adapted to cells after six blind passages. The outer capsid VP2 has been mapped the specific amino acids Q253 and A284 that were involved in cell culture adaptation of IBDV. The objective of this study was to generate a cell-adapted IBDV using RNA polymerase II reverse genetic strategy with sequences of vvIBDV-TWN as templates. pVAX/SegA/HDR and pVAX/SegB/HDR plasmids were constructed to contain Segment A and Segment B of vvIBDV-TWN strain, respectively and a hepatitis delta virus ribozyme (HDR) was added into the 3' end of each construct. A pVAX/mutSegA/HDR was also generated by introducing direct mutations (Q253H and A284T) into the VP2 protein. DF-I cells were transfected with various combinations of constructed plasmids with/without co-infected with bursa-derived vvIBDV one day after transfection. The obvious cytopathogenic effects (CPE) were only seen in the cells transfected with the combinations including pVAX/mutSegA/HDR and co-infected with vvIBDV. Cells with CPE had positive green immunofluorescence signals by indirect immunofluorescence with anti-IBDV polyclonal antibody. The products of RT-PCR from cells with CPE could be cut by restriction enzyme BglII which is the genetic tag in pVAX/mutSegA/HDR to distinguish recombinant virus from vvIBDV-TWN strain. The results indicated that the co-infection of pathogenic bursa-derived vvIBDV-TWN contributed to the adaptation in cell culture of IBDV generated by RNA polymerase II reverse genetics strategy in vitro.
URI: http://hdl.handle.net/11455/66351
其他識別: U0005-1708201117252000
Appears in Collections:微生物暨公共衛生學研究所

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