Epitope analysis and construction of a broad spectrum single chain Fv antibody against potyviruses

• Main Authors: Han-lin Liu, 劉漢麟
• Other Authors: Ya-Chun Chang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/01754119751161290946

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 97 === 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.