Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31805
標題: 以矮南瓜黃化嵌紋病毒載體在矮南瓜上表現胡瓜嵌紋病毒單鏈抗體變異區
Expression of the single-chain variable fragment of the monoclonal antibody to Cucumber mosaic virus by Zucchini yellow mosaic virus vector in squash
作者: 戴君惜
Tai, Chun-Hsi
關鍵字: single-chain variable fragment
矮南瓜黃化嵌紋病毒
Zucchini yellow mosaic virus
Cucumber mosaic virus
virus vector
monoclonal antibody
載體
單鏈抗體變異區
胡瓜嵌紋病毒
單株抗體
出版社: 植物病理學系
摘要: 在植物細胞內或細胞外表現抗體或抗體片段,以調節其所對應的抗原功能稱之為免疫調節(Immunomodulation)。這些抗體片段包括抗原結合片段(antigen binding fragment, Fab)及單鏈抗體變異區(single-chain variable fragment, scFv)可用轉基因植物的方式在植株內有效率的被表現,以調節植株的代謝途徑及使植物得到對病原菌的抗性。近年來,病毒載體系統已發展完備,可在植物體內表現外來的基因,故本實驗則是利用本實驗室已構築好的矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)重症型(p35ZGFPhis-3)及輕症型病毒(p35ZGAChis-3)為載體,攜帶胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)單鏈抗體變異區,以及本實驗室分離自洋香瓜的胡瓜嵌紋病毒鞘蛋白基因。此scFv可辨識胡瓜嵌紋病毒的鞘蛋白以及胡瓜嵌紋病毒的鞘蛋白基因,以對應轉譯的方式插入ZYMV載體之協同性蛋白(HC-Pro protein)N端,並加入組胺酸標定(histidine tag)及NIa蛋白裂解酶切位序列,使scFv能以游離方式存在。構築完成的重組體分別為p35ZCMVsf (重症型病毒攜帶CMV scFv);p35ZACCMVsf (輕症型病毒攜帶CMV scFv)。ZCMVsf來自p35ZCMVsf,ZACCMVsf來自p35ZACCMVsf,在矮南瓜上造成的病徵,都比原本的病毒載體造成的病徵輕微,且上位葉病徵會回復。ZCMVsf (由p35ZCMVsf所複製出的病毒)與ZACCMVsf(由p35ZACCMVsf所複製出的病毒)分別感染的罹病植物可經由RT-PCR及西方墨點法可證實重組病毒帶有scFv。被感染的矮南瓜上位葉及下位葉的病毒力價以ELISA測定其吸光值差距約1倍,再分別於一片上或下位葉挑戰接種CMV。觀察4-36天,接種ZGAC後(ZYMV輕症型重組病毒帶有GFP, p35ZGAChis-3), 46% (15/32棵)的植物沒有出現CMV的病徵, ZACCMVsf 及ZCMVsf 保護的植物在挑戰接種CMV於上位葉後,並不能提供顯著的保護效果;但是挑戰接種於下位葉後,只在初期提供延遲發病的效果,之後以ZACCMVsf保護者會100% (32/32 棵) 發病;以ZCMVsf保護者仍有16% (5/32棵)的植物未發病。很顯然地,植物對CMV具抗性與CMV scFv的表現無關,而是由ZGAC所提供的抗性。再則由ZYMV TW-TN3 (重症野生型)及ZGFP(ZYMV重症型重組病毒帶有GFP, p35ZGFPhis-3)保護的植物,挑戰接種CMV利用ELISA偵測判讀結果。挑戰接種上位葉,以ZYMV TW-TN3保護過的植物有19%未測到CMV,以ZGFP保護的植物則有36%。挑戰接種下位葉時,分別為25%及42%未測到CMV,所以,病毒量及GFP相信都會是影響因子之一。
The strategy called immunomodulation, describes the approach to express antibodies and antibody fragments inside and outside plant cells for modulation of the function of a corresponding antigen. The antibodies or antibody fragments, such as antigen binding fragments (Fab) and single-chain variable fragments (scFv) expressed by the transgenic approach in plants were found to effectively modulate plant metabolism and pathogen resistance. In past few years, many plant viral vectors which could express foreign proteins in planta have been developed. In this study, the plant virus vector Zucchini yellow mosaic virus (ZYMV), which was previously constructed by our laboratory, was used to express a scFv fragment against a common epitope on the coat protein of Cucumber mosaic virus (CMV) in zucchini squash. The CMV scFv fragment was in frame inserted into the severe strain ZYMV vector p35ZGFPhis-3 and the mild strain ZYMV vector p35ZGAChis-3, both of them with additional six histidine residues and NIa protease cleavage sites to process the free form proteins, to generate the recombinants p35ZCMVsf (severe) and p35ZACCMVsf (mild), respectively. On squash plants, the virus ZCMVsf (severe) derived from p35ZCMVsf produced milder symptoms of yellow mosaic and less leaf distortion than those induced by the original ZYMV severe strain. The ZACCMVsf (mild) virus derived from p35ZACCMVsf produced systemic symptoms of mild mottling on lower leaves, and then recovered on upper leaves, similar to those induced by the original ZYMV mild strain. Expression of the CMV scFv fragment from the ZCMVsf and ZACCMVsf in the infected plants was confirmed by reverse transcription- polymerase chain reaction (RT-PCR) and the free form of the protein was detected by western blotting using the monoclonal antibody against the six histidine residuses. The ZCMVsf or ZACCMVsf infected plants did not show significant degrees of protection when challenged with CMV on an upper leaf with mild mosaic. Whereas, when the challenge inoculation was applied on a lower leaf with conspicuous symptoms, the results indicated that 54% of a total of 32 ZGAC (the virus derived from p35ZGAChis-3) protected squash plants showed a significant delay (4-36 days) in symptom development and 46% of a total of 32 protected plants did not show any CMV symptoms. Also, most of the ZACCMVsf and ZCMVsf protected squash plants showed delay in symptom development at early stages, but at the later stages all of squash plants protected with ZACCMVsf showed 100% (32/32 plants) of severe CMV symptoms while 16% (5/32 plants) of the squash plants protected with ZCMVsf did not show severe CMV symptoms. Apparently, the protection against CMV was provided by the mild ZGAC virus and not related to CMV scFv in infected cells. The squash plants were first protected by ZYMV TW-TN3 (severe strain wild type) and ZGFP (derived from p35ZGFPhis-3, a severe strain carrying GFP), and then challenged CMV that were monitored by ELISA thereafter. When challenged on an upper leaf, 19% of the plants protected by ZYMV TW-TN3 and 36% protected by ZGFP were CMV negative. When challenged on lower leaf, 25% of the plants protected by ZYMV TW-TN3 and 42% protected by ZGFP were a CMV negative. The possible mechanisms of the protection provided by ZYMV vectors against the heterologous CMV are discussed.
URI: http://hdl.handle.net/11455/31805
Appears in Collections:植物病理學系

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