東亞百香果病毒單株抗體與分子檢測方法之研發及應用

百香果種苗繁殖及銷售是整個百香果產業的重要經濟利基，臺灣本土每年種苗需求量約為90萬株，其餘種苗多外銷往東南亞，年出口量達800萬株。為了防止病害隨著種苗外銷而蔓延，無病毒健康種苗的生產及病毒檢測極為關鍵。病毒危害對百香果的栽培影響巨大，其中又以東亞百香果病毒 (east asian passiflora virus, EAPV) 造成的百香果木質化病最為嚴重，受感染的植株會有生長勢低落、葉片嵌紋、果實木質化、畸型化、果腔縮小、果汁風味變差⋯⋯等病徵。相較於RT-PCR (reverse transcription-polymerase chain reaction)，利用免疫學原理之酵素連結免疫吸附法 (enzyme-linked immunosorbent assay, ELISA) 為一成本較低、效率較高的病毒檢測方法。因此，本研究針對EAPV製備高敏感度及高專一性之單株抗體，以應用於病毒免疫檢測。將EAPV之鞘蛋白基因選殖至表現載體pET-28a(+)，並以大腸桿菌系統 (Escherichia coli BL21 (DE3)) 表現重組鞘蛋白作為免疫抗原，注射小鼠產生免疫反應，經細胞融合技術，篩選獲得一單元抗體細胞株mAb7H8G9。經間接酵素連結免疫吸附法 (indirect-ELISA) 分析，純化後之抗體對EAPV病葉粗萃取液的力價可達16384倍，並且能夠檢測到稀釋640倍的病葉粗萃取液。藉由田間百香果病毒調查，檢視所設計之ELISA檢測方法的效力，顯示mAb7H8G9對臺灣主要栽培品種台農一號的檢測正確率高。另外，也針對EAPV之AO strain與IB strain都具有的保守區域以及歧異度較大的區域，分別設計出EAPV廣效性引子對，以及兩strain各自的專一性引子對，並首次建立EAPV RT-qPCR (real-time RT-PCR)。本研究研發不同面向的檢測方法，可提供未來學術研究與百香果防檢疫之應用。

關鍵字

東亞百香果病毒；單株抗體；酵素連結免疫吸附法；重組鞘蛋白

並列摘要

The propagation and exportation of seedlings have high economic value in the passion fruit industry. The domestic demand for seedlings was about 900 thousand annually in Taiwan, and approximately 8 million seedlings were exported to Southeast Asia. Given that the number of seedlings is enormous, the production and indexing of virus-free seedlings are important to prevent the spread of virus diseases via seedlings. In Taiwan, the passionfruit woodiness disease (PWD) caused by east asian passiflora virus (EAPV) is the most serious problem among several virus diseases of passion fruit. EAPV could induce mosaic and distortion symptoms on leaves, severe woody symptoms on fruits, and an unpleasant flavor of the juice. Compared to the RT-PCR (reverse transcription-polymerase chain reaction) assay, immunological detections such as ELISA (enzyme-linked immunosorbent assay) are the more economical and efficient virus-indexing methods. This study, therefore, developed the monoclonal antibody (mAb) against EAPV with high specificity and sensitivity for further immunological tests. The coat protein (CP) gene of EAPV was first cloned into the expression vector pET-28a(+). Inducing by IPTG, the recombinant CP was expressed and purified from Escherichia coli BL21 (DE3) and then immunized BALB/c mouse. After cell fusion and screening, a hybridoma cell line, 7H8G9, was obtained. The indirect-ELISA showed that the purified monoclonal antibody 7H8G9 (mAb7H8G9) could be diluted to 16384 times, and it was still effective in identifying the EAPV-infected leaf crude extracts, which was diluted to 640 times. The ELISA designed in this study was validated by field samples tests, which indicated that the detection accuracy on Taiwan’s main passion fruit cultivar, Tainung No. 1, was high. In addition, EAPV universal primer and strain-specific primers for two different strains (AO strain and IB strain) were designed and the RT-qPCR (real-time RT-PCR) assay for EAPV was established for the first time. Both immunological tests with our developed mAb7H8G9 and nucleic acid-based tests with designed primers and the probe could be reliable choices for EAPV detections, and it would be helpful for future academic research and passion fruit quarantine.