張雅君Chang, Ya-Chun臺灣大學:植物病理與微生物學研究所劉漢麟Liu, Han-linHan-linLiu2010-05-112018-06-292010-05-112018-06-292009U0001-0808200918063300http://ntur.lib.ntu.edu.tw//handle/246246/181959海芋病毒性病害在田間多為複合感染，為了減少花費與縮短檢測時間，可選擇研發同時檢測多種病毒的方法。本實驗室針對危害海芋的芋頭嵌紋病毒(Dasheen mosaic virus)、蕪菁嵌紋病毒(Turnip mosaic virus)、海芋嵌紋病毒(Zantedeschia mosaic virus)與海芋微嵌紋病毒(Zantedeschia mild mosaic virus)，利用其鞘蛋白中保守性區域製備抗原，並篩選出廣效性單株抗體(C4)。除了上述病毒外，C4抗體也可與Potyvirus屬的其他病毒反應。為瞭解C4抗體與不同Potyvirus屬病毒辨識能力之差異，我們利用噬菌體展現胜肽庫(phage display peptide library)來尋找C4抗體所辨識之抗原決定基(epitope)。首先將噬菌體胜肽庫利用C4抗體進行三次親和性篩選(panning)後，逢機挑取溶菌斑(plaque)培養，以其上清液進行間接式與競爭型 ELISA，經篩選後共挑選23個噬菌體表現株(phage clone)進行定序。將解序結果與五種 potyvirus之鞘蛋白胺基酸序列進行比對，發現胺基酸164~175 與 178~189 兩個位置可能為抗原決定基所在。由罹病植物Western blot分析結果可排除164~175位置為抗原決定基之可能性。噬菌體表現株Western blot結果顯示，若具有 Tryptophan (W)、 Leucine (L)、Glycine (G)、Glutamic acid (E)/Glutamine (Q)、Valine (V)/Isoleucine (I)或Tyrosine (Y)/Phenylalanine (F)等胺基酸，與C4抗體結合效果較佳。將Western blot與之前序列比對的結果相互對照，可推測potyvirus鞘蛋白之中WV(T)MMDGXXQV(I)EY(F)為C4抗體所辨識之抗原決定基。此外，我們嘗試構築C4之單鏈抗體(single chain Fv antibody fragment, scFv)，此重組抗體可利用蛋白質表現系統快速且大量生產抗體。首先以專一性引子對增幅出VH與VL片段，大小分別為342 bp與336 bp，並利用連接子(linker)連接成完整的scFv。利用pET表現系統得到約30.26 kDa大小之單鏈抗體，但單鏈抗體會在大腸桿菌內聚集成內含體(inclusion body)，須經過重新摺疊(refolding)過程才能恢復活性，但目前所嘗試的方法獲得之單鏈抗體會與健康植物反應。因此我們利用 Pichia 表現系統表現外泌之水溶性單鏈抗體，大小約為29.73 kDa，以1% methanol誘導96小時表現量為最高。取上清液進行透析和純化後，將純化之水溶性單鏈抗體進行dot blot與Western blot分析其專一性，結果顯示單鏈抗體在dot blot可專一地辨識PVY感染的植物樣品，而不會與健康植物有非專一性反應。Western blot結果顯示單鏈抗體具有廣效性，且與C4單株抗體辨識相同的抗原決定基。未來將利用已構築之植物表現載體表現C4單鏈抗體，觀察單鏈抗體是否能影響Potyvirus屬病毒鞘蛋白的摺疊(folding)，因而使病毒無法正確組裝，而使植物具有抗病毒能力。Mixed infection of viral disease happens frequently in calla lily field. Detection methods simultaneously targeting multiple viruses should be developed in order to save the time and cost. We cloned and expressed the conserved region of the coat protein of calla lily-infecting potyviruses including Dasheen mosaic virus, Turnip mosaic virus, Zantedeschia mosaic virus and Zantedeschia mild mosaic virus as the antigen to produce the monoclonal antibody (mAb). A broad spectrum mAb (C4) against the potyviruses was screened. It could detect at least 10 potyviruses in addition to these four potyviruses. To clarify different binding ability of potyvirus to C4 mAb, phage display peptide library was used to determine the epitope reacting to C4 mAb. After three round of panning, 38 plaques were randomly selected for test and the supernatant of phage culture was analyzed by phage ELISA and competition ELISA. Twenty-three phage clones were sequenced and their sequences were aligned with those of five potyviruses. From the result of sequence alignment, there were two possible locations of the epitope (164 to 175 and 178 to 189). According to the Western analysis of virus-infected plant samples, the possibility of location 164 to 175 could be excluded. The result of phage Western blot indicated if the phage clone contained residue W, L, G, E/Q, V/I or Y/F, its affinity to C4 mAb increased. The amino acid sequence alignment of nine phage clones and potyviruses revealed that the epitope recognized by C4 mAb was WV(T)MMDGXXQV(I)EY(F). Furthermore, we constructed a broad spectrum C4 single chain Fv antibody fragment (C4 scFv) and used prokaryotic and eukaryotic expression systems to express the recombinant antibody. The variable regions of heavy chain and light chain which are 342 and 336 bp long were separately amplified by specific degenerate primers. The VH and VL fragments were assembled with linker DNA. The 30.26-kDa C4 scFv was expressed by pET29a(+) vector in E. coli BL21 (DE3) but it formed inclusion body. The refolded C4 scFv analyzed by ELISA and Western blot showed non-specific reactions to healthy plants. In order to avoid the problem of forming inclusion body, a Pichia expression system was used to express C4 scFv. The Pichia expression vector expressed a 29.73-kDa soluble scFv due to its secretion signal peptide. SDS-PAGE analysis showed the highest expression level of C4 scFv was at 96 hr after induced by 1% methanol. The specificity of the purified C4 scFv was analyzed by dot blot and Western blot. The result showed that C4 scFv could specifically bind to the epitope of potyvirus as C4 mAb. In the future, we will express C4 scFv in plants to evaluate its effect on potyvirus particle assembly.目錄試委員會審定書......................................Ⅰ 謝..................................................Ⅱ文摘要..............................................Ⅲbstract..............................................Ⅴ、前言...............................................1、前人研究...........................................3 一、 彩色海芋與病害簡介............................3 二、 抗體的結構與功能..............................5 三、 單鏈抗體之應用................................7 四、 噬菌體表現系統................................9amp;#21442;、材料與方法........................................12 一、抗原決定基分析 (Epitope mapping)..............12 (一)親和性篩選(BioPanning)....................12 (二)測定噬菌體效價(titer).....................13 (三)噬菌體ELISA (Phage ELISA).................13 (四)競爭型ELISA (Competition ELISA)...........14 (五)抗原決定基之定位分析(Location epitope)....15 (六)蛋白質電泳(SDS-PAGE)凝膠製備..............15 (七)Phage樣品製備及SDS-PAGE電泳分析...........16 (八)染病植物樣本及SDS-PAGE電泳分析............16 (九)Coomassie brilliant blue R-250染色法......16 (十)免疫轉漬分析(Western blot)................17 (十一)植物全RNA之抽取.........................18 (十二)反轉錄聚合酶連鎖反應....................18 (十三)聚合酶連鎖反應..........................18 (十四)鞘蛋白二級結構預測......................19 二、製備與表現廣效性單鏈抗體......................19 (一)C4單鏈抗體建構策略........................19 (二)融合瘤細胞全RNA抽取.......................20 (三)mRNA純化..................................20 (四)反轉錄聚合酶連鎖反應......................21 (五)聚合酶連鎖反應............................21 (a)擴增 Heavy chain variable region.......21 (b)擴增 Light chain variable region.......22 (六)回收PCR產物之DNA片段......................22七)DNA片段黏合.......................................23八)轉型反應..........................................23九)轉型株之篩選......................................23十)菌液PCR(Colony PCR)...............................23十一)質體之小量製備(minipreparation).................24十二)重鏈/輕鏈變異區與連接子接合反應.................24a)重鏈與連接子接合反應...............................24 (b)輕鏈與連接子接合反應...........................25十三)單鏈抗體組裝反應................................25、大腸桿菌表現載體之構築............................26一)大腸桿菌表現載體之構築............................26二)單鏈抗體之誘導表現及純化..........................26a)最適誘導時間測試...................................26b)單鏈抗體純化.......................................27三)單鏈抗體重新摺疊(refolding).......................28a)逐步透析(Stepwise dialysis, SWD)...................28b)直接透析(Direct dialysis, DI)......................28四)單鏈抗體定量......................................29五)測試E. coli表現之C4單鏈抗體之專一性...............29a) ELISA.............................................29b)免疫轉漬分析.......................................30、Pichia pastoris表現載體之構築.....................30一) Pichia pastoris表現載體之構築....................30二)Pichia pastoris勝任細胞之製備.....................31三)基因重組Pichia pastoris菌株之轉型.................31四)Pichia pastoris菌株表現單鏈抗體之分析.............32a)最適誘導時間測試...................................32b)單鏈抗體純化.......................................33五)測試Pichia pastoris表現單鏈抗體專一性.............33a)免疫墨點反應法.....................................33b)免疫轉漬分析.......................................34、結果............................................. 35、C4單株抗體所辨識之抗原決定基之分析(Epitope mapping)...35 (一)Phage ELISA與競爭型 ELISA之結果...........36二)Epitope位置之分析.................................37三)Phage clone之Western blot結果.....................38四)罹病植物樣本之RT-PCR與Western blot分析結果........38五)Potyvirus鞘蛋白二級結構預測結果...................38、製備與表現廣效性單鏈抗體..........................39一) C4單鏈抗體建構策略...............................39二)融合瘤細胞全RNA與mRNA之純化.......................39三)擴增重鏈變異區(Heavy chain variable region, VH)...40四)擴增輕鏈變異區(Light chain variable region, VL)...41五)重鏈/輕鏈變異區與連接子之接合反應.................41六)單鏈抗體組裝反應..................................41 三、大腸桿菌表現載體之構築與表現..................41 (一) 大腸桿菌表現載體之構築...................41 (二)單鏈抗體之表現與純化......................42 (三)以ELISA測試E. coli表現之單鏈抗體之專一性..43 (四)以Western blot測試E. coli表現之單鏈抗體之專一性..44 四、Pichia pastoris 表現載體之構築與表現..........44 (一) Pichia pastoris表現載體之構築............44 (二)單鏈抗體之表現與純化....................................................45 (三)以dot-blot測試Pichia表現之單鏈抗體之專一性..46 (四)以Western blot測試Pichia表現之單鏈抗體之專一性..46、討論..............................................48、參考文獻..........................................55、表................................................65、圖................................................67application/pdf6288015 bytesapplication/pdfen-US噬菌體展現胜肽庫抗原決定基potyvirus單鏈抗體包含體噬甲醇酵母菌表現系統Phage display peptide libraryepitopescFvinclusion bodypichia pastoris expression systemhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181959/1/ntu-98-R96633018-1.pdf