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一種葡萄風信子病毒之鑑定及其血清與分子檢測工具之製備

Identification of a Grape hyacinth-infecting Virus and the Production of Its Serological and Molecular Detection Tools

摘要


具輕微黃化嵌紋病徵之進口葡葡風信子罹病組織粗汁液可與Potyvirus屬病毒專一性單株抗體產生正反應。進一步萃取葡葡風信子罹病組織之全量核酸,利用Potyvirus屬之廣效性引子對HRP 5/Oligo-dT進行反轉錄-聚合酶鏈鎖反應(Reverse transcription-polymerase chain reaction, RT-PCR),可增幅出一與預估相符合的核酸片段(約1.2 kb)。此RT-PCR產物經選殖及定序後,獲得一核酸Ma2選殖株。Ma2選殖株之核苷酸與胺基酸序列與Potyvirus屬的葡萄風信子嵌紋病毒(Muscari mosaic virus,簡稱MMV,序號EU042752)的鞘蛋白基之核苷酸和胺基酸序列皆有高於98%之相同度,顯示此葡萄風信子病毒爲MMV之一分離株(isolate)。進一步將Ma2選殖株之全長度鞘蛋白序列選殖於表現載體pET28b(+)上,轉型於E. coli Rosetta(DE3)宿主內並大量誘導其表現蛋白,分子量估計約29.7 kDa。經免疫分析證實此表現蛋白可與市售之Potyvirus專一性抗體反應,且將此表現蛋白經兔免疫注射後,得到對應Ma2鞘蛋白抗原之抗血清(As-Ma2)。As-Ma2抗血清可應用於酵素連結免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)、西方轉漬法(western blotting),及SDS免疫擴散反應(sodium dodecyl sulfate immunodiffusion)與同源抗原產生反應,也於ELISA反應中與其他數種potyviruses之病葉組織有正反應之結果,進一步以西方轉漬法證明As-Ma2抗血清與四種豆類病毒(BCMV、BICMV、BYMV及SMV)、兩種天鵝絨病毒(OrMV及PtVY)、兩種瓜類病毒(MVbMV及ZYMV)以及一種茄科病毒PVMV等之鞘蛋白分子量相關位置有免疫反應,顯示As-Ma2於血清反應中可廣效應用於此等potyviruses的血清檢定。利用Ma2已知序列所設計之Maup/Madw引子對,於RT-PCR反應中可於葡萄風信子罹病組織中專一性地檢測出病毒反應,但不與其他種potyviruses罹病組織有反應,此引子對可應用於MMV之專一性檢測。國外已記錄之MMV首見見於台灣,本研究針對MMV病毒之檢測試劑進行開發,對於進口葡萄風信子種球之病毒監測具有實質之助益。

並列摘要


Grape hyacinth (Muscari spp.) is an imported bulb ornamental crop mostly from the Netherlands. Chlorosis and mosaic symptoms were observed on the newly emerging leaves of some bulbs. A potyvirus can be detected from the crude saps of infected leaves by the Potyvirus-specific monoclonal antibody in enzyme-linked immunosorbent assay (ELISA). A cDNA fragment with a predicted length of 1.2 kb can be amplified by using the degenerate primers (HRP5/Oligo-dT) specific for potyviruses in reverse-transcription polymerase chain reaction (RT-PCR). The amplified cDNA fragment (Ma2) has been cloned and sequenced, and compared to those of other potyviruses available in the GenBank. The percentage of the nucleotide and deduced amino acid sequence identities of the full- length coat protein (CP) of Ma2 are higher than 98% to that of Muscari mosaic virus (MMV, Acce. No. EU042752), indicating the virus associated with grape hyacinth chlorosis and mosaic is an isolate of MMV The full-length CP gene was amplified by RT-PCR and constructed into the bacterial expression vector pET28b(+), and then transformed into E. coli Rosetta (DE3) cells for large scale protein expression. An expected 29.7-kDa over-expressed fusion protein (Ma2-CP) was purified and used as an immunogen to prepare polyclonal antiserum (As-Ma2). Antiserum against Ma2-CP was effective on detecting the homologous and heterologous potyviruses, including BCMV, BICMV, BYMV, SMV, OrMV, PtVY, MVbMV, ZYMV and PVMV, in ELISA and western blotting. The designed Maup/Madw primer pair was specifically applied on the detection of MMV isolates in grape hyancith tissues by RT-PCR. MMV is first reported in Taiwan. Our results revealed that MMV-Ma2 antiserum can be applied to detect various potyviruses with its broad-spectrum serological properties, and the primer pair (Maup/Madw) can be applied to specifically detect MMV from the imported grape hyancith bulbs based on its specific molecular identities.

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