Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36028
標題: 以竹嵌紋病毒衛星核酸載體表現日本腦炎病毒外套膜蛋白第三區域
Expression of the Domain III of Japanese Encephalitis Virus Envelope Protein with BaMV satellite-Based Vector in Plants
作者: 陳明絹
Chen, Ming-Juan
關鍵字: sat BaMV vector;竹嵌紋病毒衛星核酸載體;expression vector;Japanese Encephalitis Virus;subunit vaccine;Envelope Protein;表現載體;日本腦炎病毒;次單元疫苗;外套膜蛋白
出版社: 生物科技學研究所
摘要: 
日本腦炎在亞洲地區是一種經蚊蟲傳播的病毒性疾病,感染人類之後可能引起中樞神經系統損傷及嚴重的併發症,甚至死亡。日本腦炎病毒是黃病毒科的一員,基因體為約11Kb的正股RNA。日本腦炎病毒的寄主範圍相當廣泛,包括豬、鳥、狗等脊椎動物。傳統的日本腦炎病毒疫苗是自感染日本腦炎病毒的鼠腦中萃取,經福馬林處理成不活化疫苗,然而這樣的疫苗生產花費昂貴、具有一定的風險且免疫時效性短。因此,研發較安全、成本低廉且有效的日本腦炎疫苗是很重要的。以植物快速、便利地表達重組的病毒免疫源,以研發各種口服疫苗是目前的趨勢之一。此口服疫苗較傳統疫苗有許多優點:包括操作方便、容易量產、成本較低。此外,口服疫苗只含有部分的病毒蛋白而非完整的病毒基因體,沒有病毒性回復的潛在隱憂。竹嵌紋病毒(Bamboo mosaic virus, BaMV)屬於馬鈴薯病毒群potexvirus group)包含一正股RNA 基因體, 5端帶有cap結構及3端poly(A) 尾端序列。有些竹嵌病毒分離株紋攜帶有衛星核酸(satellite RNA, satBaMV),可表現一非結構性蛋白(P20)。由於此衛星核酸的轉譯區非複製所必須,可被外源基因取代以發展成表現外源蛋白的載體系統。本實驗中,將日本腦炎病毒外套膜蛋白的第三區域置換入衛星核酸的轉譯區,與竹嵌紋病毒一起接種於N. benthamiana以表現此日本腦炎病毒抗原蛋白。並爲比較表現外源蛋白的效率,設計數種構築體,包括包含P20蛋白或其C端部分胜肽與否(pCBSJ、pCBSJ-f50aa與pCBSJ-P20)。各重組衛星核酸與竹嵌紋病毒共同接種後,以北方漬染法及西方免疫偵測法觀察各構築體的複製和轉譯效率。另一方面,也將日本腦炎外套膜蛋白第三區域轉殖植物染色體中,經由竹嵌紋病毒感染轉殖植物後,觀察到帶有日本腦炎抗原基因的衛星核酸卡匣轉錄體比未感染的植物提高至少1000至4000倍,每克葉片可轉譯200 ng的蛋白質量。本實驗證實轉殖植物的衛星核酸卡匣上的日本腦炎抗原基因可經由竹嵌紋病毒感染而大量表現,以此新方式研發日本腦炎次單元疫苗,將可克服過去單純轉殖植物抗原蛋白表現量不足的難題。

Japanese encephalitis (JE) caused by the Japanese encephalitis virus (JEV) is spread by infected mosquitoes in Asia. It can affect the central nervous system and cause severe complications, even death. JEV, a member of the Flaviviridea family, is a single-stranded, positive-sense RNA virus with a genome size of approximately 11Kb. The JE vaccine currently used is a mouse brain-derived, formalin-inactivated JEV. This JE vaccine has many disadvantages. Thus, the development of JE vaccine that is safer, less costly and effective is important. A promising strategy for developing subunit vaccines involved in the expression of recombinant vaccine immunogens in edible plants, which can be grown and produced easily. There are many advantages in this oral vaccine than the conventional vaccine. Bamboo mosaic virus (BaMV), a member of the potexvirus, contains a single-stranded, positive sense RNA genome with a 5' cap structure and 3' poly (A) tail. A satellite RNA (satBaMV) associated with BaMV is a linear RNA molecular of 836 nts [excluding the poly (A) tail] encoding a non-structure protein of 183 amino acids (P20). The BaMV and satBaMV vector systems have been developed to express foreign genes by replacing the ORF of satBaMV. In this study, the coding sequence of domain III of JEV envelope protein cloned into the satBaMV vector can express in N.benthamiana inoculated with the helper virus infectious cDNA clone pCB. Various constructs were designed to compare the efficiency of expression, such as the constructs either fusion with c terminus of P20 or 50 amino acids of P20 c-terminus or not (ie. pCBSJ, pCBSJ-f50aa, and pCBSJ-P20). After inoculation of plants with pCB and each recombinant satellite construct, the replication of chimeric satellite and translation of protein were determined by Northern and Western blot analyses. Besides, the JEV gene was introduced into plants by Agrobacterium transformation. After challenge with BaMV, the transcripts of satellite cassette containing the sequence of the domain III of JEV E protein were amplified. Expression level of the domain III of JEV envelop protein induced by BaMV infection was significantly high than transgene expression.
URI: http://hdl.handle.net/11455/36028
Appears in Collections:生物科技學研究所

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