Potyvirus廣效性單株抗體辨識之抗原決定基之分析與單鏈抗體之構築

Abstract

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.