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標題: | 利用諾羅病毒拆開之P蛋白質自我組裝形成P顆粒呈現抗原 Presenting Antigens in Norovirus P Particle Formed by Automatically Assembled Split P Protein |
作者: | Yin-Hsi Lin 林映希 |
指導教授: | 黃慶璨 |
關鍵字: | 諾羅病毒,疫苗平台,嗜甲醇酵母菌,外鞘P蛋白質,抗原呈現, Norovirus,antigen,P particle,vaccine platform,Pichia pastoris, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 諾羅病毒外鞘P蛋白質具有三個表面環狀結構適合作為抗原嵌入位。嵌有外來抗原的P蛋白質組裝成P粒子後,可大幅提高外來抗原的免疫原性,是一個近年來被廣泛研究,極具潛力的疫苗平台。更重要的是P蛋白質可以利用大腸桿菌或嗜甲醇酵母菌Pichia pastoris進行大量生產奠定了產業化的基礎。然而,現今以兩端固定在P蛋白質上的方式呈現抗原,卻可能因外來抗原的結構特殊,或體積龐大而破壞P蛋白質本身的結構,進而阻礙P粒子的正常組裝,這可能就是現在以諾羅病毒做為多抗原呈現平台時,多以呈現小片段胜肽為主的原因。因此,本研究嘗試將P蛋白質由第2個環狀結構的胺基酸位置拆成兩段(分別為PN及PC),分別利用兩個甲醇誘導的啟動子各自表現後,利用SDS-PAGE及西方墨點法進行胞內或胞外蛋白質的生產確認,再搭配蛋白質原態電泳、Ni-NTA pull down及Ni-NTA純化及膠體過濾法證實拆開的P蛋白質(PN及PC)可以重新組裝成完整的P蛋白質,更可以進一步組裝成P粒子。將來,利用拆開的P蛋白質呈現外來抗原時,可藉由一端游離的形式攜帶抗原,降低抗原大小及種類對P蛋白質結構造成的壓力,使諾羅病毒P粒子多抗原呈現平台更具彈性及應用性。 As a vaccine platform, norovirus P particle not only can present antigens in a highly repetitive manner to enhance the immunogenicity of the foreign antigen, but also have three surface loops for antigens insertion in a P protein. Moreover, P protein can be produced in many host systems including E.coli and Pichia pastoris. However, the most common way to present whole protein antigen with two terminus linked to P protein might disrupt the structure of P protein, and consequently lead to the difficulty of P particle assembly. This might be the cause of difficulties of presenting large antigen or antigen with spatially distant N and C termini. In order to overcome this obstacle, P protein was split into two parts from surface loop 2, so that antigen was presented with only one terminus fused, to decrease the conformational stress on P protein. Here we showed that the split P proteins (PN and PC) were successfully produced by P. pastoris and can be scaled-up by a 5-L fermenter. Ni-NTA pull-down assay, native PAGE and gel filtration data together showed that PN and PC interact with each other and could form multimers. The establishment of this split P protein vaccine platform might improve the flexibility of presenting different antigens. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71888 |
DOI: | 10.6342/NTU201804136 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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