感染鳶尾花引起嵌紋病徵之Potyvirus病毒鑑定及利用細菌表現病毒鞘蛋白製備病毒多元抗體

Abstract

本研究於日本進口之鳶尾花植株發現黃化嵌紋病徵之葉片組織，以ELISA (Enzyme-linked immunosorbent assay)檢測出與Potyvirus屬病毒專一性抗血清產生正反應，進一步純化罹病葉片之全量核酸，利用Potyvirus屬之廣效性引子對HRP 5/Oligo dT進行反轉-聚合酶鏈鎖反應(Reverse transcription-polymerase chain reaction, RT-PCR)，將預期1.3-kb核酸產物進行選殖及定序後，獲得一核酸選殖分離株JI 2。JI2分離株與GenBank上已登錄之鳶尾花嵌紋病毒(Butterfly flower mosaic virus，簡稱BFMV，序號AM774001）之鞘蛋白核苷酸與氨基酸序列相同度(identities)均人於99.6%，推測屬於相同病毒之不同系統。設計可增幅JI2全長度鞘蛋白基因之專一性引子對，經RT-PCR增幅後，將其選殖於表現載體pET28b (+)上，再轉型於E. coli Rosetta (DE3)宿主內誘導大量表現蛋白之生成，將分子量約29.5 kDa之表現蛋白經兔免疫注射後，得到對應JI2之多元抗體（#162）。此多元抗體可應用於ELISA、西方轉漬法(Western blotting)，及SDS免疫擴散反應(Sodium dodecyl sulfate immunodiffusion)與同源抗原產生專一性反應。針對JI2之已知核酸序列所設計之JI-up/JI-dw引子對，利用RT-PCR可專一性地檢測出感染BFMV之鳶尾罹病組織，此等自製以JI2分離株為來源所製備對應鳶尾花嵌紋病毒引子對及抗體，可實際應用於進口鳶尾花種球之病毒監測，提供了我國對此病毒之自主檢測能力之實質助益。 Iris (Iris japonica), native in Japan and Mainland Chain, is a bulb floral crop imported to Taiwan. Several iris plants exhibited yellowing mosaic symptoms on the leaves emerging from the bulbs were recently observed. The infected leaves exhibiting symptom was positively reacted to the Potyvirus-specific monoclonal antibody (Agdia Inc.) in enzyme-linked immunosorbent assay (ELISA). A predicted 1.3 kb DNA fragment was amplified from the symptomatic leave tissues by use of reverse-transcriptase polymerase chain reaction (RT-PCR), with the use of potyvirus-degenerate primers. An isolate (JI2) was cloned and sequenced from the RT-PCR amplicons and compared to those of other plant viruses available in the GenBank. The percentage identities of the nucleotide and amino acid sequences of the full-length coat protein (CP) gene of JI2 were both higher than 99.6% comparing to the Butterfly flower mosaic virus (BFMV, GenBank Acc. AM774001). It indicated that JI2 isolate is probably a strain of BFMV. The full-length of JI2-CP gene was subsequently amplified and constructed into the expression vector pET28b (+), then transformed into E. coli Rosetta (DE3) cells. An expected 29.5-kDa overexpressed fusion protein was purified and used as an immunogen to prepare polyclonal antiserum. The designed JI-up/JI-dw primer pair was applied in the detection of BFMV in iris tissues by RT-PCR. The prepared antiserum (As-JI2) was proved to be useful in ELISA, western blotting and SDS-immunodiffusion tests for the detection of BFMV in virus-infected iris samples. The As-JI2 was suitable to be applied on the BFMV detection for the imported iris bulbs.