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研究生:吳純宜
研究生(外文):Chun-I Wu
論文名稱:感染彩色海芋之蕪菁嵌紋病毒之分子特性分析及抗體製備
論文名稱(外文):Molecular characterization and antibody preparation of a Turnip mosaic virus isolate infecting calla lily
指導教授:張雅君張雅君引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:74
中文關鍵詞:彩色海芋單元抗體蕪菁嵌紋病毒
外文關鍵詞:monoclonal antibodyZantedeschiaTurnip mosaic viruscalla lily
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彩色海芋為台灣新興花卉之一,由於花形獨特且花色變化多,廣受消費者喜愛,為最具潛力之球根花卉。但栽培過程極易遭受病毒病害侵襲,造成經濟損失。2002年,自田間取得一複合感染之彩色海芋葉片組織,將其汁液接種於指示植物白藜上,結果出現褪綠單斑病徵。經三次單斑分離並回接彩色海芋組培苗,出現黃色斑點及條斑病徵;以間接酵素連結免疫分析,顯示其與anti-potyvirus之抗體呈正反應;以陰染法染色,於電子顯微鏡下亦可觀察到絲狀病毒顆粒。由於本實驗室所研究感染彩色海芋的potyvirus,如芋頭嵌紋病毒、彩色海芋嵌紋病毒及彩色海芋微嵌紋病毒,寄主植物多侷限在天南星科植物,故推測可能是異於前三者之另一potyvirus。為了更進一步獲得序列資料以鑑定此病毒種類,以針對Potyvirus屬所設計之廣效性引子對(degenerate primers)進行RT-PCR,得1.7 kb片段。經選殖及解序等工作,取得部份NIb至3’端非轉譯區之序列,與NCBI資料庫進行比對,確認此一病毒為危害十字花科甚為嚴重的蕪菁嵌紋病毒(Turnip mosaic virus, TuMV),並將此病毒分離株稱之為TuMV-ZAN。進一步進行全長度基因體序列之解序,得全長序列共9834個核苷酸,包含5’及3’端非轉譯區及P1、HC-Pro、P3、6K1、CI、6K2、NIa、NIb及CP等基因。2004年再於田間分離到另一感染彩色海芋的蕪菁嵌紋病毒,將其3’端之基因選殖並定序,發現其CP基因與TuMV-ZAN只有89%相同度,因此命名為TuMV-ZAN2分離株。將這兩個分離株鞘蛋白基因與來自其他國家的蕪菁嵌紋病毒分離株進行類緣分析,結果顯示這兩個分離株分別落在world-B及basal-BR group中。
為方便將來蕪菁嵌紋病毒之學術研究及田間檢測,將此一病毒之鞘蛋白基因選殖至表現載體pET-29a(+),並以大腸桿菌系統(Escherichia coli BL21 (DE3))之表現重組鞘蛋白作為免疫抗原,生產抗體。所得之抗血清以間接酵素連結免疫分析及西方轉漬法測試抗體力價及專一性,結果抗血清力價至少達25000倍,並且只與TuMV有專一性反應。此外,也將此類表現蛋白免疫注射小白鼠,經融合瘤等技術篩選到一單元抗體細胞株,稱之為Mab403。以間接酵素連結免疫分析,發現這個單元抗體不只對蕪菁嵌紋病毒之鞘蛋白有反應,亦可廣效地偵測到至少另外八種potyvirus。
A calla lily leaf sample infected by several viruses was collected from the field in 2002. When Chenopodium quinoa was inoculated by the leaf extract, it appeared chlorotic local lesions. After three successive single lesion isolation, a virus isolate was obtained. The yellow spot and stripe symptoms showed when the isolate was transferred back to tissue-cultured transplants of calla lily. The virus isolate reacted positively with the monoclonal antibody specific against potyvirus group by indirect enzyme-linked immunosorbent assay (ELISA). Flexuous virus particles about 750 nm in length were observed by the transmission electron microscopy. All of above evidences revealed that the isolate is a potyvirus but different from those calla lily-infected potyviruses studied in our laboratory, including Dasheen mosaic virus (DsMV), Zantedeschia mosaic virus (ZaMV) and Zantedeschia mild mosaic virus (ZaMMV). To further characterize the viral molecular nature, a pair of degenerate primers designed according to the conserved sequences of the reported potyviruses was used to amplify 1.7 kb fragment by RT-PCR. After cloning and sequencing, viral genomic sequences from partial NIb gene to 3’UTR were obtained and they are similar to Turnip mosaic virus (TuMV) by the database alignment. Therefore, this virus isolate was named as TuMV-ZAN. Using the same clonging strategy, we obtained the full length genomic sequences of TuMV-ZAN including 5’ and 3’ UTR, P1, HC-Pro, P3, 6K1, CI, 6K2, NIa, NIb and CP genes. Another TuMV isolate infecting calla lily was found and sequenced in 2004, but its nucleotide sequences of CP was only 89% identity with those of TuMV-ZAN. This isolate was named TuMV-ZAN2. Phylogenetic analysis of the CP gene indicates that the ZAN and ZAN2 isolates are located at world-B and basal-BR group, respectively.
In order to obtain the antiserum of TuMV for further study and field survey, we cloned the CP gene of TuMV-ZAN into pET-29a(+) expression vector. After transformation to Escherichia coli BL21 (DE3) and induction by IPTG, the recombinant CP of TuMV-ZAN was expressed and purified to immunize rabbits. The titer and specificity of the prepared TuMV antiserum was tested by indirect-ELISA and western blot analysis. Besides, we used the same kind of expressed CP to immunize mouse and obtained a hybridoma cell line-Mab403 after screening. We found that the monoclonal antibody (Mab403) could reacte not only with TuMV but with at least eight other potyviruses.
中文摘要………………………………………………………………………. 1
英文摘要………………………………………………………………………. 3
壹、前言………………………………………………………………………. 5
貳、前人研究………………………………………………………………… 7
參、材料與方法……………………………………………………………....15
一、病毒來源與分離………………………………………………………15
二、生物特性分析…………………………………………………………15
(一) 寄主範圍測試……………………………………………………...15
(二) 間接酵素連結抗體免疫吸附法(Indirect-ELISA)………………...16
(三) 以電子顯微鏡觀察病毒顆粒……………………………………...17
(四) 病毒顆粒之純化…………………………………………………...17
三、分子特性分析………………………………………………………..18
(一) 植物全RNA之抽取……………………………………………….18
(二) 病毒核酸之抽取…………………………………………………...18
(三) 反轉錄聚合酵素連鎖反應(RT-PCR)……………………………...19
(四) 基因選殖、定序及分析……………………………………………20
(五) 序列比對及分子類緣關係分析(phylogenetic analysis)…………..24
四、多元抗體之製備……………………………………………………..25
(一) 重組鞘蛋白質體之構築…………………………………………...25
(二) 重組鞘蛋白之誘導表現及純化…………………………………...25
(三) 蛋白質電泳分析(SDS-PAGE)……………………………………..26
(四) 多元抗體之生產…………………………………………………...27
(五) 以西方轉漬法及indirect-ELISA測試多元抗體之專一性及力價.27
五、單元抗體之製備……………………………………………………...29
(一) 動物免疫…………………………………………………………...29
(二) 細胞融合…………………………………………………………...29
(三) 細胞保存法………………………………………………………...31
(四) 單株抗體之生產…………………………………………………...32
(五) 免疫球蛋白之純化………………………………………………...32
(六) 抗體力價之測定…………………………………………………...33
肆、結果………………………………………………………………………34
一、生物特性分析………………………………………………………...34
(一) 彩色海芋病徵及寄主範圍測試之結果…………………………...34
(二) 利用Indirect-ELISA檢測之結果…………………………………35
(三) 病毒顆粒純化與電子顯微鏡觀察之結果………………………...35
(四) 病毒鞘蛋白分子量測定…………………………………………...35
二、分子特性分析………………………………………………………..35
(一) 基因體3’端定序與分析之結果…………………………………...35
(二) TuMV-ZAN全長度基因體序列之解序結果………………………36
(三) TuMV分離株之核酸序列比對與類緣分析之結果……………….37
三、多元抗體之製備……………………………………………………...38
(一) 重組鞘蛋白基因質體(pET-TCP)之構築………………………….38
(二) 重組鞘蛋白之誘導表現…………………………………………...39
(三) 以西方轉漬法及indirect-ELISA測試多元抗體力價及專一性….39
四、單元抗體之製備……………………………………………………..40
(一) 抗原之種類與來源………………………………………………...40
(二) 單元抗體株之獲得………………………………………………...40
(三) 測試單元抗體細胞株力價之結果………………………………...40
(四) 測試Mab403與其他potyvirus反應之結果………………………40
伍、討論……………………………………………………………………...42
陸、圖表……………………………………………………………………...46
柒、參考文獻………………………………………………………………...63
捌、附錄……………………………………………………………………...70
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