- 學位論文
轉導重組桿狀病毒於不同哺乳動物細胞最適化條件之研究及開發新型重組桿狀病毒之牛流行熱疫苗
Optimization of Environmental Factors in Different Mammalian Cells for Transduction of Genetic Recombinant Baculoviruses into Various Mammalian Cell Lines and Development of a BEFV G Subunit Vaccine by a Novel Baculovirus Display System
摘要
目前桿狀病毒 (baculovirus) 已被認為是種新型載體可提供基因治療和產製重組蛋白於昆蟲與哺乳動物細胞中。在本研究著重於將未濃縮的桿狀病毒提供最適化的轉導條件將基因送入各種哺乳動物細胞內。以本實驗室已構築之pBacCE載體接上綠螢光蛋白 (enhanced green fluorescent protein, EGFP) 基因，使其能在哺乳動物細胞內表現綠螢光蛋白，並且再將水泡性口炎病毒之醣蛋白 (vesicular stomatitis virus G protein, VSV G) 基因建構於重組桿狀病毒之p10 啟動子之後，稱本構築載體為pBacVCE。發現以未濃縮的pBacVCE重組之桿狀病毒在D-PBS的溶液下感作4個小時的轉導，於大部分的細胞株，皆能得到多數細胞受到轉導並表現出綠螢光蛋白。接著將外源真核蛋白-家禽流行性感冒病毒 HA 之膜蛋白基因於 pBacVCE 載體中，此載體命名為 pBacVE-HA2 。以西方墨點分析其蛋白的表現量，以評估及建立最佳之轉導條件。而桿狀病毒呈現系統於哺乳動物細胞能大量表現真核蛋白，以作為日後應用於開發疫苗或基因治療的工具之一。另一方面，將牛流行熱之醣蛋白 (bovine ephemeral fever virus G protein, BEFVG) 構築於重組桿狀病毒呈現載體，因其外套膜之醣蛋白具中和性抗體決定位，可以有效的刺激牛隻產生保護性免疫反應，可作為疫苗開發。在本研究進行建構桿狀病毒呈現載體具有四對啟動子與G蛋白基因，以加強BEFVG 蛋白的產量，本研究之重組桿狀病毒載體以具有四對啟動子之病毒，所得之BEFV G蛋白產量最高預期能得到較高效能的免疫性反應。在未來會依循轉導之最適化條件與開發新型的重組桿狀病毒達到高表現量、高效率、低成本的次單位疫苗與有用之重組蛋白。
並列摘要
Baculovirus is a novel vector that as a tool of gene therapy, and provides a useful system for expression of recombination protein in insect and mammalian cells. In this study, we established a transduction protocol using a novel baculovirus display system for efficient gene delivery into various cell lines as a tool in developing subunit vaccines or gene therapy tools. We used newly developed vector pBacCE that including a reporter gene, enhanced green fluorescent protein (EGFP) gene under the CMV promoter and polyhedron promoter, and then clone vesicular stomatitis virus (VSV) G protein gene under the p10 promoter called pBacVCE. Next HA of the membrane protein of avian influenza virus (AIV-HA) constructed to pBacVCE vector, called pBacVCE-HA2. The pBacVCE-HA2 applies to transduction protocol. The HA protein, a foreign protein expressed in mammalian cells, we assay the protein by Western blotting. We have successfully established a novel expression vector and transduction protocol for high level of protein expression in various mammalian cell lines when the genetic recombinant baculovirus transduction occurred at 25℃ for 4 h by using Dulbecco’s phosphate-buffered saline (D-PBS) as the transduction solution. The bovine ephemeral fever virus (BEFV) G protein contains type-specific and neutralizing antigenic sites that can induce protective immunity in cattle. Therefore, we also construct a genetic recombinant baculovirus expressing the bovine ephemeral fever virus (BEFV) G protein for producing subunit vaccine for BEF. To achieve high level expression of BEFV G protein, we have also constructed a novel expression vector that includes quadruple promoters for expressing the BEFV G gene to enhance the production of BEFV G protein. Further, the novel recombinant baculovirus was provided with high level of BEFV G protein by Western blotting. Taken together, these date indicate that the baculovirus vector includes quadruple promoter for expressing BEFV G protein should be a useful subunit vaccine that has some characteristics about high expression, high efficient and lower cost.