瓜類褪綠黃化病毒多元抗體之製備及其應用

Production of Polyclonal Antiserum Against Cucurbit Chlorotic Yellows Virus and Its Application for Diagnosis

"台灣地處亞熱帶，氣候相當適合瓜類栽種，瓜類作物遂成為台灣重要的經濟作物之一。我國每年瓜類作物的栽種面積大約有23,000公頃以上，主要栽培區域以台南市、嘉義縣、雲林縣為主。病害是瓜類作物生長的限制因子之一，其中包括病毒、細菌及真菌等所引起之病害。2009年在雲林地區發現大量瓜類作物出現黃化現象，經研究發現與2004年發生於日本的瓜類褪綠黃化病毒（Cucurbit chlorotic yellows virus, CCYV）所引起的病害相同，此病毒病害已造成瓜類作物嚴重危害。選殖CCYV病毒鞘蛋白（coat protein, CP）基因以供製造抗血清之用。根據選殖株pGEMT-CP/DH5α基因定序之結果得知鞘蛋白基因之核酸長度為735 bp，此基因轉譯成蛋白質時可產生245個胺基酸且其分子量為28 kDa。為大量生產鞘蛋白，將鞘蛋白基因重組至選殖株pQE31-CP/M15中，蛋白質表現分析證實此選殖株可在pQE系統中穩定表現，經大量純化後也成功製造出鞘蛋白之抗血清，可實際應用於CCYV之免疫磁珠檢測分析。"

"The subtropical climate in Taiwan is favor for growing cucurbits, which are economically important. The annual planted area of cucurbit crops is about 23,000 ha in Taiwan and mainly in Tainan, Chiayi and Yunlin. Occurrences of diseases, including viruses, bacteria and fungi, are the limiting factors for cucurbit cultivation. In 2009, a virus-like disease causing chlorosis and etiolation symptoms on melon was discovered at Yunlin County. The causal agent was further identified as Cucurbit chlorotic yellows virus (CCYV), which was first reported in Japan in 2004. This virus has become an important threat of cucurbit production in Taiwan. In this study, the coat protein (CP) gene of CCYV was cloned and expressed for producing antiserum. The clone pGEMT-CP/DH5α carrying the full-length CCYV CP gene verified by sequencing was obtained. The length of CCYV CP gene is 735 bp to encode a 245 aa protein with a molecular weight of 28 kDa. Subsequently, the CCYV CP gene was cloned to a bacterial expression vector to obtain the clone pQE31-CP/M15. Protein expression analysis confirmed that the CCYV CP gene can be stably expressed in the bacterial pQE system. The antiserum against the CCYV CP was produced. Our result showed that the produced antiserum can be applied in immunomagnetic bead-captured reverse transcription-polymerase chain reaction for detection of CCYV in field cucurbit samples."