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dc.contributor.authorCheng-Hao Huangen_US
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dc.description.abstractDengue is one of the most important vector-borne viral diseases in humans. During the egress of virion particle formation after dengue viral infection, pre-membrane (prM) and envelope (E) protein associate into heterodimers at ER membranes and prM protein is cleaved by the trans-Golgi resident furin protease to form the M envelope protein and the soluble pr segment, which is released into the extracellular medium upon particle secretion. prM cleavage marks maturation of flavivirus virions, which intimately correlated to change of conformation of envelope protein complexes and is a prerequisite for E dimerization. Therefore, there is an urgent need to develop antibodies to gain insight into how the interaction between prM and E affect the particle formation and secretion from host cells. The aim of this study was to (1) develop an anti-M antibody from mouse sera immunized by recombinant M protein produced from E coli; and (2) investigate the virus-like particle (VLP) maturation by modulating the furin cleavage site and using the anti-M antibody for detection. The prM (18.1–19.1 kDa) is a precursor to the M structural protein (7–9 kDa), which contains two-thirds of C-terminus as membrane anchor domain. In order to develop an anti-M only antibody, the full-length M sequence of DENV-2 was cloned into pET22b and the recombinant M protein was purified by nickel column under denature condition. The sera from mice immunized three times with recombinant M protein with Freud's adjuvant was applied to detect M protein from cells transfected by VLP-producing plasmids. According to the results of immunofluorescent assay (IFA) and western blotting, our data suggested that the anti-M protein antibody could detect M protein intra-cellularly and extra-celluarly secreted VLPs. Two forms of the M protein could be detected in our study including the prM in the immature virions and M protein in extracellular mature virions. Interestingly, by modulating the furin cleavage site, we could increase the maturity of VLPs by up to 90%. In conclusion, our study highlighted the importance of the furin-cleavage site in modulating the maturation and particle secretion of dengue VLP formation.en_US
dc.description.abstract登革病毒是現今人類社會重要的蟲媒介病毒疾病之一。受病毒感染後,病毒在複製的過程中前體膜蛋白 (prM) 和套膜蛋白 (E) 會在內質網中形成二聚體,隨後prM蛋白會被運送至高基氏體中受到furin酵素酶的作用,進而形成膜蛋白(M) 以及親水性的pr蛋白片段,並且隨著病毒顆粒釋放出細胞外。黃病毒顆粒上的prM切割位是決定E蛋白是否可以順利改變構型成為二聚體並成為成熟病毒顆粒至關重要的因素。因此,目前迫切需要開發出足以觀察prM和E蛋白之間交互作用的抗體,並且藉此抗體去探討在宿主細胞中病毒顆粒的形成及分泌。 本研究的目的如下 (1) 藉由大腸桿菌所表現之重組M蛋白免疫小鼠,並從血清中取得抗M蛋白抗體; (2) 利用抗M蛋白抗體觀察在furin切割位上的點突變所產出的類病毒顆粒之成熟度。 PrM蛋白 (18.1-19.1 kDa) 是M結構蛋白 (7-9kDa) 的前體蛋白,其C'端約三分之二為固定於膜上的跨膜結構。為了開發出一個抗M蛋白的抗體,將登革二型中全長的M蛋白構築到pET22b載體上,並且利用鎳離子管柱純化出重組的疏水性M蛋白。接著將純化後的蛋白與弗氏佐劑混合,以肌肉注射的方式對小鼠進行三次免疫,最後取其血清用以觀察轉染質體至細胞後所得的類病毒顆粒。 根據免疫螢光反應以及西方轉漬法的結果,我們的數據顯示,抗M蛋白抗體可以偵測到細胞內及細胞位的類病毒顆粒,並且同時可以偵測到在未成熟病毒顆粒上的prM,以及在成熟病毒顆粒上的M蛋白。有趣的是,我們透過在furin切割位上的突變結果,可以將類病毒顆粒之成熟度提升至90%。 總之,透過本實驗可得知furin切割位上的突變對於類病毒顆粒的成熟度及分泌能力的重要性。zh_TW
dc.description.tableofcontents第一章、 文獻回顧 1 1.1 登革病毒簡介 1 1.2 登革病毒結構及其基因體 1 1.3 登革病毒生活史 2 1.4 登革病毒E及prM / M蛋白之結構特性及功能 3 1.5 登革病毒prM/E蛋白之間的交互作用 4 1.6 類病毒顆粒 (Virus-like particles, VLPs) 6 1.7 登革病毒膜蛋白抗體發展及應用 6 1.8 蛋白表現系統之比較 7 1.9 實驗動機與目的 9 第二章、 材料與方法 10 2.1 登革病毒M結構蛋白原核表現質體之構築 10 2.2 重組結構蛋白之表現 15 2.3 登革furin切割位序列突變及病毒顆粒生產 21 第三章、 結果 27 3.1 登革病毒M結構蛋白原核表現質體之構築 27 3.2 登革病毒pET22b-M重組蛋白表現 28 3.3 登革furin切割位序列突變及病毒顆粒生產 30 第四章、 討論 33 第五章、 參考文獻 36 第六章、 附錄 43zh_TW
dc.subjectMutant at plasmiden_US
dc.titleDevelopment of anti-M antibody to investigate the interaction of pr, M and E protein during dengue virus-like particle formationen_US
dc.typeThesis and Dissertationen_US
Appears in Collections:微生物暨公共衛生學研究所


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