Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36291
標題: 應用竹嵌紋病毒載體表現疫苗蛋白
Application of BaMV based-vector for vaccine production
作者: 陳宗賢
Chen, Tsung-Hsien
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;chimeric virus particles;vaccine;plant bioreactors;重組病毒顆粒;疫苗;植物生物反應器
出版社: 生物科技學研究所
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摘要: 
植物病毒可在植物中進行複製並能經構築發展成為載體系統,所生產的重組病毒顆粒,具有發展成為疫苗的潛能。本實驗利用竹嵌紋病毒載體,以不同方式表現不同重組病毒顆粒。首先,本研究利用竹嵌紋病毒載體的抗原呈現系統(epitope presentation system),將高致死性傳染性華氏囊病毒(very virulent Infectious bursal disease virus)的抗原直接構築在竹嵌紋病毒的外鞘蛋白N端,命名為pBIBD2。接種具感染力的重組竹嵌紋病毒株質體 (pBIBD2)於葉片時,可穩定的將高致死性傳染性華氏囊病毒的抗原表現在BIBD2重組病毒外鞘蛋白,並組裝在BIBD2重組病毒顆粒表面。雛雞經由肌肉免疫注射BIBD2的重組病毒顆粒後,雞隻可產生抗傳染性華氏囊病毒抗體及有效保護高致死傳染性華氏囊病毒的感染。其次,本研究將111個胺基酸的日本腦炎抗原 (EDIII)構築於重組竹嵌紋病毒外鞘蛋白N端,但無法獲得重組病毒顆粒。因此,利用可在轉譯過程發生部分裂解的口蹄疫病毒2A序列,連接EDIII與外鞘蛋白,構築質體pBJ2A。將具感染力的pBJ2A質體,接種於植物後,經西方免疫標定結果顯示,接種植株產生EDIII與EDIII2ACP二種重組蛋白,並可組裝成BJ2A重組病毒顆粒。利用日本腦炎EDIII抗體的免疫金標定顯示,EDIII可呈現於BJ2A重組病毒顆粒表面上。小鼠經腹腔免疫BJ2A重組病毒顆粒後,可產生日本腦炎病毒的抗體,並可中和日本腦炎病毒的感染。最後,為測試外源胜肽的特性對重組病毒的表現之影響。本研究將竹嵌紋病毒載體在外鞘蛋白的N端融合了不同來源與特性的外源胜肽,檢視這些重組病毒在植物中的感染力與累積量。結果顯示,這些外源胜肽的N端所具有的親水性、等電點與電荷,C端的二級結構,和片段的大小會影響重組病毒的感染力與累積量。本研究證實,利用竹嵌紋病毒載體可在植物中產生攜帶傳染性華氏囊和日本腦炎抗原的重組病毒顆粒。並且證實外源胜肽影響重組病毒感染力與累積量的因子,可提升重組病毒顆粒表達外源胜肽的效率與重組病毒顆粒疫苗的應用。此外,竹嵌紋病毒經修飾後,可表現更大片段蛋白於重組病毒顆粒上,優於其他植物病毒的限制。綜上所述,本研究探討外源蛋白表面抗原影響竹嵌紋病毒重組顆粒及累積之可能因子,並以實例證實竹嵌紋病毒載體系統極具潛力可發展為疫苗生產技術平台。

Plant viruses can be modified genetically to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as a vaccine candidate. In this study, Bamboo mosaic virus (BaMV)-based vector was used to explore different expression strategies for recombinant CVPs. Firstly, we demonstrate the potential for vaccine development of BaMV epitope presentation system, where the antigen from very virulent Infectious bursal disease virus (vvIBDV) was fused to the N-terminus of BaMV CP. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV antigen on its CP. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV challenges. Secondly, we produce the BaMV CVPs, by fusing a sequence encoding 111 amino acids of Japanese encephalitis virus (JEV) EDIII at the N terminal of BaMV CP, but the infectivity of this chimera was severely impaired. Therefore, the cotranslational dissociation sequence of Foot-and-mouth disease virus (FMDV) 2A peptide was inserted at the junction of EDIII and viral CP to facilitate partial cleavage during polyprotein processing. The recombinant BaMV, pBJ2A, was thus constructed to enable the generation of recombinant virus, BJ2A, when inoculation onto the plants. Western blot analyses showed EDIII was expressed as the free-form and EDIII2ACP fusion proteins in infected plants. As revealed by immunoelectron microscopy, EDIII could be presented on the surface of BJ2A. After intraperitoneal injections of the purified BJ2A, mice produced antibodies against JEV and exhibited neutralizing ability against JEV infection. Finally, factors significant in determining the infectivity and accumulation level of given chimeras were examined using different constructs harboring foreign proteins of various lengths, physical/chemical properties, and sources. The infection induced by these constructs revealed important features of foreign protein: i) the hydrophilicity, isoelectric point, and N terminus charge, ii) the secondary structure in the C terminus, and iii) length sizes effects on the chimeras infected and accumulated. Therefore, this study demonstrates the production of effective CVPs vaccine candidates against IBDV, and JEV in plants by BaMV-based vectors. Furthermore, the BaMV-based CVPs may meet the requirement of expressing longer foreign peptides. In conclusion, this study demonstrated the high potential of BaMV-based vector system as a platform for production of vaccine candidates, and analyzed the properties of foreign proteins affecting the assembly and accumulation of chimeric viruses, which could be applied in future development of BaMV-based foreign protein expression systems.
URI: http://hdl.handle.net/11455/36291
其他識別: U0005-1906201216312100
Appears in Collections:生物科技學研究所

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