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標題: | 以SARS-CoV之DNA及重組腺病毒疫苗免疫小鼠並利用大腸桿菌系統表現SARS-CoV套膜蛋白 Immunization of mice with DNA- and recombinant adenovirus-based SARS-CoV vaccines and expression of SARS-CoV envelope protein via E. coli system |
作者: | Yen-Chun Lin 林彥君 |
指導教授: | 楊雅倩 |
關鍵字: | 嚴重急性呼吸道症候群,冠狀病毒,重組腺病毒疫苗, SARS-CoV,coronavirus,recombinant adenovirus vaccine, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 嚴重急性呼吸道症候群(severe acute respiratory syndrome,SARS)為一具有高度傳染性和高致死率的新興疾病,在2002年至2003年間造成全球性的流行。研究發現引起SARS的病原為一新種的冠狀病毒,稱作SARS-associated coronavirus (SARS-CoV)。臨床研究顯示,SARS病患可產生抗病毒結構蛋白的中和性抗體,加上目前並無有效對抗SARS的治療方法,因此發展預防性的SARS疫苗至為迫切。
重組腺病毒可攜帶並有效表現外來基因,是目前廣泛應用的疫苗系統。本論文的主要研究方向是以Balb/c小鼠的動物模式,測試帶有SARS病毒基因的DNA與重組腺病毒疫苗。重組腺病毒(Adv/SME)疫苗的設計是於腺病毒載體上攜帶三種SARS-CoV結構蛋白質基因:棘蛋白(spike protein,簡稱S)、膜蛋白(membrane protein,簡稱M)以及套膜蛋白(small envelope protein,簡稱E)基因。本研究的第一部份即是製備DNA與重組腺病毒兩種疫苗,並在293A細胞測試DNA疫苗之病毒基因表現。首先以大量製備的方式純化出質體DNA疫苗(pShuttle/SME),並進行質體DNA轉染,將pShuttle/SME送入293A細胞,利用反轉錄-聚合酶鏈鎖反應與西方墨點法,分別偵測三種病毒基因的mRNA與蛋白質表現。結果發現S、M、E基因在293A 細胞內皆有mRNA的表現,但無法以西方墨點法測得蛋白質的表現。另外,將Adv/SME以293A細胞大量複製並純化,取得高效價的重組腺病毒疫苗之後,對八週大的Balb/c小鼠進行疫苗施打,爲比較不同施打策略誘發之免疫反應,施打方式分為兩種:(一) 單次施打重組腺病毒疫苗,稱為one-shot接種;(二) 先施打三劑DNA疫苗,每劑相隔兩週,四週後再追加一劑重組腺病毒疫苗,稱為prime-boost接種。施打疫苗過程中於特定時間點採集小鼠血清,分析其抗體反應。 為偵測免疫小鼠的血清抗體,以評估疫苗所引發免疫反應之成效,論文第二部分乃利用大腸桿菌表現GST-E融合蛋白質(fusion protein),先分別以抗E蛋白之兔子抗血清與抗GST蛋白之小鼠單株抗體確定GST-E蛋白質於大腸桿菌中的表現量,之後利用其為抗原來源,以西方墨點法偵測免疫小鼠的抗體反應。觀察one-shot接種組的小鼠血清,發現免疫後並未測得專一性的抗體反應;而prime-boost接種組的免疫後血清,雖然出現抗體反應,但比較實驗組與對照組的結果,兩者並無顯著差異。 The severe acute respiratory syndrome (SARS) is a newly emerged disease with high infectivity and morbidity, which caused massive outbreak worldwide from 2002 to 2003. The causative agent for SARS has been identified as a novel coronavirus named SARS-associated coronavirus (SARS-CoV). Clinical trials on SARS patients demonstrated that these people developed neutralizing antibodies against virus structural proteins. To date there is no efficient treatment for SARS, so the development of preventive vaccines is urgently necessary. Recombinant adenovirus (ADV) is a widely used viral vector system and is known for effective delivery of foreign genes and high-level protein expression. In this study, we immunized Balb/c mice with DNA and recombinant adenovirus-based SARS vaccines. We used the recombinant ADV,Adv/SME,as vaccine which contains three of SARS-CoV structure protein genes, spike (S), membrane (M) and envelope (E). At the first part of this study,we prepared DNA and recombinant adenovirus vaccines, and evaluated the gene expression of DNA vaccine in 293A cells. First of all, the plasmid DNA vaccine,pShuttle/SME,was purified by massive preparation. Reverse transcription-polymerase chain reaction and western blotting were carried out to detect the expression of S, M and E genes in 293A cells transfected with pShuttle/SME. The mRNA expression of three genes was detectable; however, respective protein expression was non-detectable via western blot assay. On the other hand, high-titer Adv/SME was amplified in 293A cells. Two immunization strategies were performed in eight-weeks Balb/c mice to compare their efficiency in inducing antibody responses. The single shot immunization of recombinant adenovirus vaccine is so-called one-shot vaccination, while three DNA vaccines priming then one recombinant adenovirus boosting is so-called prime-boost vaccination. Collection of mouse sera was accompanied with the vaccination regimen. For preparing antigen sources to detect antibody responses of immunized mice,the GST-E fusion protein was expressed in E. coli and was confirmed by western blotting. The GST-E fusion protein could be detected by rabbit anti-E antisera and mouse anti-GST monoclonal antibody. Then we measured the antibody responses of immunized mice via western blotting using GST-E fusion protein. It was shown that no specific anti-E antibody was detected after one-shot vaccination. Similarly, there was no significant differences between experimental and control sets after prime-boost vaccination, either. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35505 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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