Occurrence of Chrysanthemum virus B in Taiwan and Preparation of Its Antibody against Coat Protein Expressed in Bacteria

菊花病毒B（Chrysanthemum virus，B，簡稱CVB）乃一分佈全球，可造成菊花減產之重要病毒。自民國89年至94年，我們針對彰化縣內各菊花種苗繁殖圃及切花生產田區進行CVB發生情形之調查。在9個不同地點共收集504株菊花葉片樣品，以間接式酵素連結免疫分析法(indirect ELISA)進行病毒偵測，結果其中有394株被偵測出感染CVB，這是台灣地區首次發現CVB感染菊花之證據。在田間偵測中我們同時發現CVB的偵測與樣品採集時間之氣溫變化具相關性；在15~20℃的低溫季節可輕易地以ELISA檢測出感染CVB之菊花，且所得之EIA值均非常明確。然而，當溫度上升至25℃以上，相同樣品之EIA值會下降，趨近於健康對照之EIA值；這些結果顯示台灣的冬季較適宜執行CVB之檢測。為了解台灣CVB分離株之特性，本試驗根據已發表之CVB鞘蛋白核酸序列設計出一組專一性引子對（CVB-up及CVB-dw），可針對菊花樣品進行RT-PCR而穩定增幅出一個含1028個核苷酸之DNA產物。該DNA序列可轉譯出一個含316胺基酸之蛋白質，且與20個登錄於GenBank之CVB鞘蛋白(CP)基因序列比對後發現其核苷酸序列相同度介於78.4-88.0%之間，而胺基酸序列之相同度則介於83.1-93.7%，證實此序列即為台灣菊花上所分離之CVB之鞘蛋白基因。為製作專一性抗體供未來CVB之偵測，本試驗遵循本研究室過去已經多次報告之策略，利用細菌表現病毒鞘蛋白之方式，將CVB之CP基因構築於細菌表達載體pET28b(+) (Novogen, Inc., Madison, WI, USA)中，再將其轉型於E.coli strain Rosetta (DE3)菌株，經由IPTG之誘導使細菌大量表達CVB之CP。應用純化之表現蛋白為抗原，已經成功製備出一株抗血清(#107)，此抗血清可應用indirect ELISA偵測感染CVB之菊花，與商用CVB抗血清(Agdia Inc. Elkhart, IN, USA)比較，感病組織在相同稀釋倍數下本策略所製備之抗血清其EIA值經常大於購自Agdia之商用抗血清，且對健康無病菊花之背景值極低，證明＃107確實為一優質之檢測抗體。

關鍵字

菊花；菊花病毒B ；表現載體；反轉錄聚合酵素連鎖反應；血清；細菌表現病毒鞘蛋白

並列摘要

Chrysanthemum virus B (CVB) is well known for its global distribution and adverse effect on chrysanthemum production. From the year of 2000 to 2005, we conducted an extensive survey for the occurrence of CVB in different chrysanthemum plantations and cutting producing nurseries in Changhwa County. Among 504 chrysanthemum leaf samples collected from 9 different locations, 394 of them were detected with CVB infection in indirect ELISA. This is the first evidence showing the existence of CVB in chrysanthemum in Taiwan. We also found that the detection of CVB by ELISA was affected by the air temperature during seasonal field surveys. In seasons when temperature between 15-20℃, CVB infected plants were readily detected by ELISA and exhibited evident ETA readings. However, the ETA readings of the same samples would drop to healthy control levels when temperature rose above 25℃. This result implies winter is the appropriate time to perform CVB indexing in chrysanthemum in Taiwan. To characterize CVB isolates from Taiwan, their CP gene was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using a set of primer (CVB-up, CVB-dw) designed in this study according to the sequences available in GenBank. A DNA product of 1028bp was amplified, cloned in pCRII-TOPO plasmid and subsequently sequenced. It was found to contain an open reading frame with 316 amino acid residues corresponding to the size of reported CVB CPs and it shared 78.4 to 88.0% and 83.1 to 93.7% identities in nucleotide and amino acid sequence, respectively, with 20 known CVB CP gene sequences documented in GenBank. In order to produce specific antiserum against CVB for its detection, we expressed the cloned CVB CP gene in bacteria culture and using bacteria expressed CP as immunogen for antiserum preparation. An antiserum (#107) was prepared against the expressed CVB CP and shown to be useful in ELISA to detect CVB in chrysanthemum plants. By comparing with a commercialized CVB antiserum (Agdia Inc. Elkhart, IN, USA), the reactivity in terms of ETA readings of antiserum #107 to the same dilution of infected chrysanthemum tissue was always higher than that of Agdia's CVB antiserum.

並列關鍵字

chrysanthemum ； CVB ； expression vector ； RT-PCR ； serology ； bacteria-expressed virus coat protein

國際替代計量

Occurrence of Chrysanthemum virus B in Taiwan and Preparation of Its Antibody against Coat Protein Expressed in Bacteria

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