海芋嵌紋病毒重組鞘蛋白抗血清之製備及其應用

The Production and Application of the Antiserum against Zantedeschia mosaic virus Recombinant Coat Protein

胡文綺(W. C. Hu) ； 林為方(W. F Lin) ； 黃偉洲(W. Z. Huang) ； 黃金興(C. H. Huang) ； 張雅君(Y. C. Chang)

《植物病理學會刊》 16卷1期 (2007/03) Pp. 31-40

https://doi.org/10.6649/PPB.200703_16(1).0004

摘要

海芋為天南星科球根花卉，因廣受大眾歡迎，已成為國際間重要的花卉作物。田間栽種的海芋植株常遭受病毒為害，近幾年來國內外的研究顯示海芋嵌紋病毒(Zantedeschia mosaic virus ZaMV)感染率遠高於芋頭嵌紋病毒(Dasheen mosaic virus, DsMV)，且罹病植物病徵明顯，嚴重影響商品價值。本研究依據ZaMV彩色海芋分離株(ZaMv-ZAN)之序列設計專一性引子對，從其cDNA株中增幅出全長度鞘蛋白基因，選殖至表現載體後，再以大腸桿菌表現ZaMV重組鞘蛋白，並且製備出ZaMV抗血清。以間接式酵素連結抗體免疫吸附法(indirect-ELISA, I-ELISA)測試，結果顯示ZaMV抗血清具有極高的力價。而免疫轉漬分析證明ZaMV抗血清不會與健康海芋、DsMV或海芋微嵌紋病毒(Zantedeschia mild mosaic virus, ZaMMV)反應，顯示其具高專一性。當以健康海芋粗萃取液對感染ZaMV的海芋粗萃取液進行5倍系列稀釋，藉此樣品測試免疫吸附反轉錄聚合醃連鎖反應(immunocapture RT-PCR, IC-RT-PCR)和I-ELISA的檢測靈敏度。實驗結果顯示當使用ZF2與ZR0引子對時，IC-RT-PCR比I-ELISA靈敏度高25倍；若改用ZF3與ZR0引子對時，可再提高IC-RT-PCR約125至625倍的效果，意即檢測靈敏度較I-ELISA高3125至15625倍。本研究所研發的ZaMV檢測方法未來可應用於海芋種苗病毒驗證作業，以協助海芋健康種苗之生產。

關鍵字

海芋 ； 海芋嵌紋病毒 ； 酵素連結抗體免疫吸附法 ； 免疫吸附反轉錄聚合酶連鎖反應

並列摘要

Calla lilies (Zantedeschia spp.), the bulbous flowers in the family Araceae, are very popular among consumers, hence they become the international important flower crops. The calla lily plants are frequently infected by viruses in the field. According to recent reports, Zantedeschia mosaic virus (ZaMV), which induces obvious symptoms and seriously affects the flower value, has much higher infection rate than Dasheen mosaic virus (DsMV). In this study, ZaMV specific primers were designed based on the sequence of ZaMV-ZAN isolate. The full-length coat protein gene of ZaMV was amplified from its cDNA clone, and then cloned into the expression vector. Recombinant ZaMV coat protein was expressed by Escherichia coli and used as antigen to prepare ZaMV antiserum. The result of indirect-ELISA (I-ELISA) demonstrated ZaMV antiserum had high titer. In addition, its specificity was confirmed by immunoblot analysis since it did not react with healthy, DsMV- or ZaMMV-infected calla lilies. To compare the detection sensitivities of immunocapture-RT-PCR (ICRT-PCR) and I-ELISA, ZaMV-infected calla lily extracts were five times diluted serially with healthy plant extracts, and then treated as testing samples. The experimental results indicated that IC-RT-PCR with ZF2 and ZRO primers was about 25 times more sensitive than I-ELISA. However, when ZF3 and ZRO primers were used, the detection limit of IC-RT-PCR times could increase 125-625 fold. In other words, the detection of IC-RT-PCR could be 3125-15625 times more sensitive than 1-ELISA. The ZaMV detection methods developed in this study can be applied to virus certification scheme of calla lily and help to produce healthy seedlings in Taiwan.

並列關鍵字

calla lily ； Zantedeschia mosaic virus ； I-ELISA ； IC-RT-PCR