利用重組鞘蛋白製備可偵測多種海芋病毒之廣效性單株抗體

Abstract

海芋(calla lily)原產於南非，其分類地位屬於天南星科(Araceae)、馬蹄蓮屬(Zantedeschia)，由於其花型獨特典雅，花色多樣，且瓶插壽命長，近年來廣受消費者喜愛，並為台灣重要經濟花卉產業之ㄧ。病毒病害為海芋栽培之限制因子，且國內外報導指出感染海芋之病毒以Potyvirus屬為最大宗，會造成植株矮化、葉面嵌紋、生長不良、花器畸型等，進而影響切花產量及產值。目前針對感染海芋的potyviruses之檢測技術，除了利用反轉錄聚合酶鏈鎖反應(RT-PCR)外，以血清學原理所發展的酵素連結抗體免疫吸附法(ELISA)偵測技術更廣為使用。為了在檢測上節省耗費並加快篩選健康海芋種苗的速度，故本研究嘗試研發可廣泛性偵測多種potyviruses之單株抗體(monoclonal antibody)。其抗原之製備是以國內曾報導感染海芋之五種potyviruses包括海芋潛徵病毒(Calla lily latent virus, CLLV)、芋頭嵌紋病毒(Dasheen mosaic virus, DsMV)、蕪菁嵌紋病毒(Turnip mosaic virus, TuMV)、海芋嵌紋病毒(Zantedeschia mosaic virus, ZaMV)及海芋微嵌紋病毒(Zantedeschia mild mosaic virus, ZaMMV)之鞘蛋白(capsid protein)基因為對象，經由序列比對，選取序列保守性較高之區域，長度為121個胺基酸，分別設計專一性引子對，以PCR增幅出目標片段，再將五種病毒之鞘蛋白保守性片段進行選殖並接合(ligation)，製備重組蛋白(recombinant protein)並以大腸桿菌表現。接著進一步純化此重組蛋白，免疫老鼠以製備單株抗體，經indirect-ELISA方式篩選所獲得之多株穩定表現之融合瘤細胞(hybridoma cells)，從中選取一反應最好之細胞株，其抗體可專一性的檢測出五種海芋病毒的鞘蛋白。將細胞注入老鼠腹腔產生大量腹水，並測試其力價為105。另外，於廣效性測試中，此單株抗體亦可偵測到至少另外10種potyviruses。因此，未來可望將此potyvirus之廣效性單株抗體，作更進一步之應用。

Calla lily originated from southern Africa belongs to the genus Zantedeschia, the family Araceae. Recently calla lily has become one of economically important flowering plants and continues to increase in popularity because of its unique shape, spathe coloration and long-term lifespan. During the cultivation of calla lily, viral disease is one of the limiting factors in Taiwan as well as in other countries. Potyviruses are the major viruses infecting calla lily and often cause symptoms of stunt, mosaic, growth decline and flower deformation. The yield and quality of cut flowers are seriously affected. At present, detection methods for potyviruses in calla lily such as reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) are commonly employed. Interestingly, ELISA is considered by the industry to be more suitable than RT-PCR as routine indexing technique of large amount of samples. To save the materials, labors and time in detection, therefore in this study, we tried to generate a monoclonal antibody which can detect as many potyviruses as possible. The highly conserved region of capsid protein (CP) gene of calla lily-infecting potyviruses reported in Taiwan including Calla lily latent virus (CLLV), Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Zantedeschia mosaic virus (ZaMV) and Zantedeschia mild mosaic virus (ZaMMV) was selected after amino acid sequences aligned. The conserved region of 121 amino acids in length was PCR amplified by specific primers of each virus. The PCR fragments were ligated to construct two kinds of expression vectors, recombinant proteins were expressed in E. coli expression system and then purified as antigens for immunization. After cell fusion, we used the expressed proteins and potyvirus-infected plant samples as antigens to screen the hybridoma cell lines by indirect-ELISA. One stable cell line secreting potyvirus-specific antibodies named as MAb C12-C4 with best reactivity and was used for ascites production. The mouse ascites from MAb C12-C4 gave well detectable reactions to antigens at dilution up to 105 times. In the spectrum test, this MAb could detect at least ten other potyviruses by indirect-ELISA. From our experimental results, MAb C12-C4 has the potential to be used for further researches and applications.