利用細菌表現康乃馨斑駁病毒之鞘蛋白製備多元抗體及其檢測應用

Abstract

彩色海芋為我國近年新興之花卉，其切花產品有外銷日本市場之實績，極其經濟發展潛力。本研究由進口自紐西蘭的海芋種球(Fandango品系)上，發現部分植株在首次抽生之葉片上出現黃色斑點癥狀，類似前人所報告之康乃馨斑駁病毒(Carnation mottle virus, CarMV)感染之病徵。因此抽取樣品之全量核酸，以包含全長度CarMV鞘蛋白基因所設計之簡併式引子對CarMV-u/5' -CAACACATTTCRATWA-3'和CarMV-d/5' -TCACATCCT ATAAACA進行反轉錄-聚合酶鏈鎖反應(Reverse transcription-polymerase chain reaction, RT-PCR)，獲得一人小約1.0 kb與預估值相符合的核酸片段。此核酸產物經選殖及定序後，所得之Fo25選殖株，其核苷酸及胺基酸序列與GenBank上已登錄之CarMV之鞘蛋白基因相同度(identities)均人於96%，證實Fo25確為CarMV的一個分離株。進一步比對分離自康乃馨與海芋之各種CarMV分離株，發現其鞘蛋白基因之核苷酸及肢基酸序列間相同度均高於96％。而類緣分析結果則顯示CarMV之不同分離株似有依寄主之不同而形成不同群組之趨勢。依據所獲之Fo25核苷酸序列，設計可增幅其全長度鞘蛋白基因之專一性引子對，經PCR增幅後將其選殖於表現載體pET28b(+)上，再轉型於E. coli Rosetta (DE3)宿主內以誘導其人量生成39 kDa之表現蛋白，經免疫分析證實此表現蛋白可與購自Agdia公司(Elkhart, Indiana, USA)之CarMV抗體反應，確實其有CarMV鞘蛋白之抗原性。將此表現蛋白經兔免疫注射後，得到對應CarMV-Fo25抗血清。此抗血清可應用於ELISA (Enzyme-linked immunosorbent assay)、西方轉漬法(Western blotting)，及SDS免疫擴散反應(Sodium dodecyl sulfate immunodiffusion)與同源抗原產生強烈專一性反應。且於應用在ELISA進行田間樣品檢測時，其反應性(reactivities)穩定高於購自Agdia之對照抗體，並旦可同時檢測出感染CarMV之海芋及康乃馨樣品，而對照之Agdia抗體則僅與康乃馨樣品有反應。此一抗體之成功製備對我國進口花卉種球或種苗之CarMV病毒監測將有實質之助益。 Calla lily (Zantedeschia spp.) is an emerging floral crop in Taiwan and considered with high economic potential. In this study, some calla lily plants bearing with chlorotic spots on the newest leaves coming from the bulbs imported from New Zealand were frequently observed. The symptom was quite similar to that caused by Carnation mottle virus (CarMV) reported by Chen et al., (2003). By the use of degenerate primers flanking complete coat protein (CP) gene of CarMV, a predicted 1.0 kb DNA was consistently amplified from the calla lily plants exhibiting chlorotic spots. An isolate (Fo25) was cloned and sequenced from the RT-PCR amplicons and compared to those CarMV corresponding sequences available in the GenBank. The percentage identities of CP gene of Fo25 in the sequences of nucleotide and amino acid were both higher than 96% comparing to those of CarMV, indicating that Fo25 isolate is a strain of CarMV. Full-length of CarMV's CP gene was subsequently amplified and constructed into the expression vector pET28b(+), and transformed into E. coli Rosetta (DE3). An over expressed protein with an expected molecular mass of 39-kDa was further purified and used as immunogen to prepare polyclonal antiserum. The produced antiserum (Fo25) was tested and found to be applicable in ELISA, western blotting and SDS-immunodiffusion tests for the detection of CarMV in field samples of calla lily and carnation. In these serological tests the reactivity and specificity of antiserum against Fo25 are found significantly better than the control antiserum purchased from Agdia Co.. Sequences of CP gene obtained from five CarMV isolates from carnations and two from calla lilies were used to study their phylogenetic relationship. Genetic analysis of the amino acid sequences of CP gene revealed that there was possibly genetic clusters among different CarMV isolates infecting different hosts.