- 學位論文
利用桿狀病毒表現系統開發犬瘟熱次單位疫苗
Development of canine distemper virus subunit vaccine using baculovirus display system
摘要
犬瘟熱病毒（Canine distemper virus, CDV）屬於副黏液病毒科（Paramyxoviridae）之麻疹病毒屬（Morbillivirus），為一種高度發病率與死亡率之接觸性傳染病。犬瘟熱經常發生在犬隻及其他食肉性動物上，包括浣熊、臭鼬及狐狸。3-6個月之年幼犬隻最容易遭受感染，且死亡率高於成犬。未經免疫之成犬同樣也屬高度感染群。犬瘟熱病毒具一單股RNA，包裹在脂質雙層蛋白中。由目前之研究報告指出，CDV外套膜蛋白中之血液凝集素 (Haemagglutinin protein, H)為CDV主要誘發中和抗體之蛋白，可達到對犬隻產生免疫之效果。本研究以桿狀病毒表現系統及本實驗室開發之新型載體(pBacSC)作為平台，以呈現病毒H蛋白於病毒封套及細胞膜上。新型載體建構有桿狀病毒跨膜區域基因(gp64 TM)、桿狀病毒細胞質區域基因(gp64 CTD)、綠色螢光蛋白基因（green florescence protein, GFP）及四個限制酶切位(Multiple cloning site)；GFP可作為篩選標記，而gp64 TM及gp64 CTD可使外源基因表現於桿狀病毒封套膜上。首先，我們將H蛋白之 ectodomain選殖至pBacSC載體，再將重組質體DNA轉形至DH10Bac E.coli並分離出重組Bacmid DNA 並轉染於昆蟲細胞(Sf9)，形成”重組桿狀病毒”，使H蛋白呈現於桿狀病毒封套膜或細胞膜上。透過西方墨點法、共軛焦電子顯微鏡及免疫金電子顯微鏡證明H蛋白成功地表現於病毒封套膜上。隨後大量收集培養的重組桿狀病毒並進行動物免疫試驗，收集老鼠血清並分析重組H蛋白在動物體內引發之免疫反應，期望可以開發犬瘟熱病毒次單位疫苗。
並列摘要
Canine distemper virus (CDV), a member of the genus Morbillivirus in the Paramyxoviridae family, causes a highly contagious disease with high morbidity and mortality. CDV occurs among domestic dogs and many other carnivores, including raccoons, skunks, and foxes. Young puppies of the ages between 3 weeks and 6 months old are most susceptible to the disease and are more likely to die when infected than adults. Non-immunized older dogs are also highly susceptible to the disease. The distemper virus consists of a single strand of RNA, encased in a protein coat which is again encased in a fatty envelop. According to previous studies among the canine distemper envelope proteins, protein H is the major antigenic determinant that induces neutralizing antibody production to achieve effective immune status in canines. In this study, a universal baculovirus surface display system (UBSDS) was used as the platform. The pBacSC was constructed with GFP as a selection marker; The new vector contained four multiple cloning sites and the gp64 TM and CTD were included for displaying the recombinant proteins. The H ectodomain has already been cloned into the reconstructed pBacSC vector and the recombinant plasmid DNA will be transformed into DH10Bac competent cells to form recombinant bacmid DNA. The recombinant bacmid DNA was transfected into insect cells, forming “recombinant baculovirus”, expressing H protein ectodomain on its envelope. Analysis by Western blot, Confocal microscopy and immunogold microscopy were carried out to confirm whether H proteins expressing on baculovirus envelope were successful. A great quantity of the recombinant baculoviruses were obtained and the recombinant baculoviruses wrer collected and injected into mice. The mouse sera were analyzed to check the mouse immune response to the recombinant H protein. The purified protein will then be used to produce canine distemper virus vaccine. This may reduce protein production and purification costs and in turn reduce canine distemper virus infection rate.