Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31878
標題: 木瓜輪點病毒P1蛋白及木瓜畸葉嵌紋病毒鞘蛋白在大腸桿菌之表現及其抗體之製備
Production of Antibodies to Bacterial Expressed P1 Proteins of Papaya Ringspot Viruses and Coat Protein of Papaya Leaf Distortion Mosaic Virus
作者: 詹淑茹
Chan, Shu-Ju
關鍵字: papaya ringspot virus
木瓜輪點病毒
PRSV
protease
bacterial expression system
inclusion body
strain specific protection
papaya leaf distortion mosaic virus
PLDMV
degenerate primers
limited dilution
蛋白裂解酵素
細菌蛋白表現系統
內含體
系統專一性保護作用
木瓜畸葉嵌紋病毒
簡併式引子對
稀釋終點法
出版社: 植物病理學系
摘要: 木瓜輪點病毒 (papaya ringspot virus, PRSV) 屬於馬鈴薯Y病毒屬 (the genus Potyvirus),其基因表現策略為產生一複合大蛋白 (polyprotein),再經蛋白裂解作用產生各具功能的蛋白產物。P1蛋白位於整個複合大蛋白的N端,具蛋白裂解酵素 (protease) 的活性,蛋白大小為63 kDa,較其他馬鈴薯Y群病毒 (potyviruses) 之P1蛋白大了18~34 kDa。PRSV的寄主範圍狹窄,但依其感染的寄主範圍不同,又可分為木瓜型 (PRSV P) 和西瓜型 (PRSV W) 二型,兩者在血清學上不能區分,僅在寄主專一性上有所不同。而P1基因是馬鈴薯Y病毒屬中變異最大的基因,故本研究以其蛋白產物製備血清,期望能區分PRSV之不同型病毒與各分離株,增加分析與偵測上之便利性。P1蛋白屬於非結構性蛋白 (nonstructural protein),在罹病植物體內存量少,且不穩定,純化不易,故採用細菌蛋白表現系統 (bacterial expression system),在細菌體中表現P1蛋白。將台灣木瓜型病毒PRSV P-YK及西瓜型PRSV W-CI完整的P1基因與其C端截斷之之蛋白 (truncated protein) 片段載入表現載體中,在Escherichia coli細菌體BL21(DE3) strain中表現蛋白。實驗結果以P1 之C端截斷之蛋白構築體表現量較佳,其在表現過程中形成大量之內含體 (inclusion body) ,且產量甚佳。收取不溶內含體之部份 (inclusion body fraction),利用膠片 (SDS-PAGE) 回收再加以純化濃縮。以回收的蛋白進行血清之製備,所得之PRSV P-YK多元抗體,能以西方轉漬法 (Western blotting) 偵測到病葉中P1蛋白之存在,並能區分夏威夷品系 (PRSV P-HA) 與臺灣品系 (PRSV P-YK及PRSV W-CI),在單元抗體的選殖中,目前所得到之細胞株系亦有相同的結果。另一方面,為調查木瓜輪點病毒不同品系在田間之複雜性,以了解系統專一性保護作用 (strain specific protection) 在木瓜輪點病的防治方法上,包括交互保護與育成病毒鞘蛋白轉基因木瓜等策略之限制。從中部各地木瓜田間分離不同病徵型之木瓜輪點病毒,卻得到一與木瓜輪點病毒無血清關係的分離株。此分離株在木瓜上造成之病徵為葉片黃化萎縮、具明顯嵌紋病徵,果實上具不連續的密集黃斑,果肉有硬塊,植株生長受阻。經鞘蛋白核酸序列比對的結果,與日本琉球Maoka等人所發表在木瓜上發現之木瓜畸葉嵌紋病毒 (papaya leaf distortion mosaic virus, PLDMV) 相近。此病毒為台灣首次發現之木瓜畸葉嵌紋病毒,推測其在田間長期被木瓜輪點病毒所掩蓋。為了解其在田間分佈之情形,儘速的製備快速的檢測工具則為當務之急,然在接種多種指示植物後,發現皆不造成感染,與琉球Maoka等人發表之PLDMV PV5分離株能在刺角瓜、矮南瓜、甜瓜等植物上造成嵌紋 (mosaic) 等病徵不同。在無適當的繁殖寄主供大量純化病毒的情況下,為快速製備血清,採稀釋終點法 (limited dilution),得一純系病毒,以簡併式引子對 (degenerate primers) 選殖出鞘蛋白基因,載入細菌表現系統中表現,同樣在不溶內含體之部份 (inclusion body fraction) 表現大量之蛋白,抽取此部份蛋白,經電泳回收並加以純化,製備血清。所得之血清,能利用酵素連結抗體法 (ELISA,enzyme-linked immunosorbent assay) 測得病葉上PLDMV之存在,並可應用在西方轉漬法 (Western blotting) 之偵測。現階段已應用於偵測PLDMV在田間分佈之狀況。
Papaya ringspot virus (PRSV) is a member of the genus Potyvirus. The PRSV genome encodes a single large polyprotein that is proteolytically processed into final products by virus-encoded proteases. The P1 protein located at N-terminus of the polyprotein functions as a protease to release itself by autocatalysis. The predicted molecular weight of PRSV P1 protein is 63 kDa, 18~34 kDa larger than other potyviruses. Although the host range of PRSV is very narrow, most isolates of PRSV are divided into two major types, type P (PRSV P) and type W (PRSV W), according to their difference in host reactions. The two type viruses are indistinguishable in serology using antibodies to coat protein (CP), cylindrical inclusion protein (CIP), and amorphous inclusion protein (AIP). Since the P1 gene is most variable in potyvirus genome, antibodies against P1 proteins may be used to distinguish different strains and types of PRSV. In this investigation, the complete P1 proteins and C-terminal truncated P1 proteins of a Taiwan P type virus PRSV P-YK and a Taiwan W type virus PRSV virus PRSV W-CI were expressed in bacterial expression system. Large amounts of truncated P1 fusion proteins were expressed as inclusion body in the insoluble cytoplasmic fraction. The inclusion bodies were collected and the target proteins were purified by SDS-PAGE. The eluted proteins were used to produce antibodies in rabbits and mice. The polyclonal antibodies to the P1 truncated proteins of P-YK and W-CI were able to detect the P1 proteins in infected Cucumis metuliferus by Western blotting, and able to distinguish the P type Hawaii strain PRSV P-HA from these two P and W type Taiwan strains. On the other hand, to avoid the effects of strain-specific protection for controlling the papaya ringspot virus disease by CP-transgenic resistance or cross protection. Different isolates of viruses infecting papaya in Taiwan were collected from fields. Unexpectedly, an isolate collected from Dalee, Taichung county, was found unrelated to PRSV in serology. This isolate caused symptoms of mosaic, yellowing and distortion on leaves, and disconnected yellowing spots on fruits. Analysis the coat protein revealed that the isolate is closely related to the previously reported PV5 isolate of papaya leaf-distortion mosaic virus (PLDMV) from Okinawa. This isolate, designated as PLDMV DL, represented the first record of PLDMV in Taiwan. To realize the distribution of PLDMV, the production of antibody as a detection tool is essential. The host reactions indicated that the isolate PLDMV DL did not infect any other plants tested except papaya, and thus considered different from the PV5 isolate of PLDMV which causes mosaic symptom on Cucumis metuliferus, C. sativus, C. melo, and Cucurbita pepo. Since there was no proper propagation host for purification of the virus, the CP of PLDMV DL was constructed in protein expressing vector to overexpress the CP in bacteria cells. The CP fusion protein was expressed in large amount and presented mostly in the insoluble cytoplasmic fraction. The inclusion body was collected and the fusion protein was purified by SDS-PAGE and used to produce the antibody in rabbits. The polyclonal antibody was able to detect PLDMV DL in infected leaves in indirect ELISA, and in Western blotting. The antibody of PLDMV was currently being used to detect the distribution and prevalence of PLDMV in Taiwan.
URI: http://hdl.handle.net/11455/31878
Appears in Collections:植物病理學系

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