Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90038
標題: Development of a novel transgenic cell-suspension culture system to produce chimeric Bamboo mosaic virus particles
建立一套新穎轉基因細胞懸浮培養系統生產嵌合竹嵌紋病毒顆粒
作者: 塔米爾
Thangarasu Muthamil Selvan
關鍵字: 竹嵌紋病毒
轉錄後基因靜默
重組病毒顆粒
細胞懸浮培養
疫苗
竹嵌紋病毒載體
Bamboo mosaic virus
post-transcriptional gene silencing
recombinant virus particles
cell-suspension culture
vaccine
BaMV viral vector
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摘要: 隨著基因工程工具與高通量篩選技術時代的來臨,帶來分子生物與生物醫學領域的重大革新,次單位疫苗的發展更取代傳統動物與細胞培養所生產的疫苗,過去20年來由植物衍生的疫苗由於便宜與方便管理是一套迷人的策略; 主要區分為穩定或暫時表現的兩套系統不同的系統各有優點與其限制轉基因植物屬於穩定表現的系統,可以持續且穩定的生產疫苗,但表現量相對暫時表現系統來的略低; 暫時性表現系統,透過融合胜肽於病毒外鞘蛋白,所形成的嵌合型病毒顆 (chimeric virus particles, CVPs) 可以迅速生產出高產量的疫苗,但由於病毒的基因體沒有插入植物基因體,因此相較於轉基因植物無法持續且穩定的生產。本研究建立一套新穎的植物懸浮細胞培養系統來生產疫苗,以竹嵌紋病毒外鞘蛋白N端融合一段來自口蹄疫病毒的病毒蛋白1 (VP1) 之抗原決定基,將此嵌合竹嵌紋病 (chimeric BaMV-CP) 與野生型竹嵌紋病毒株 (wild-type BaMV) 之cDNA轉殖Nicotiana benthamiana,利用轉殖株葉片建立懸浮培養的轉基因菸草細胞,利用西方墨點法與酵素結合免疫吸附分析 chimeric BaMV-CP 與 wild-type BaMV 的表現量,結果顯示轉基因懸浮培養細胞株 BdT38 與 BdT19每20克懸浮細胞可以分別得到1.5 與2.1 mg 的嵌合竹嵌紋病毒顆粒 (CVPs), 免疫電子顯微鏡中VP1 抗原決定基存在於嵌合竹嵌紋病毒顆粒的表面上,施打嵌合竹嵌紋病毒顆粒於天竺鼠可有效產生中合性抗體。為了簡化純化嵌合竹嵌紋病毒顆粒,我們發展一套利用分子篩層析取代傳統離心的純化病毒的原型流程,結果顯示此方式可有效從菸草懸浮細胞回收到嵌合竹嵌紋病毒。我們研究考量經濟效益,而發展出一套以植物作為生產免疫胜肽 (immunopeptide) 疫苗的新平台,並嘗試簡化純化病毒的步驟,以增加疫苗大規模生產的效率。
Advent of genetic engineering tools and high-throughput screening technologies have revolutionized in the field of molecular biology and biomedical science. As a consequence, these technologies enabled the development of subunit vaccines to replace traditional animal and bacterial cell-cultures based vaccines. Similarly, last two decades plant derived antigens have been a fascinating strategy to use as vaccines candidates due to low cost and convenient administrations. Production of vaccine antigens in plants is often achieved through stable or transient expression systems. However, these technologies have certain advantages and limitations; transgenic plants can produce vaccine antigens consistently but yield is very low, in case of transient expression, in which the fused peptide expresses on the chimeric virus particles (CVPs) combined with rapid and high yield production in whole-plants system but not consistent due to viral genome is not integrated into the plant genome. In this study, a novel vaccine production method is developed using plant cell suspension culture system. We utilized this unique transgenic cell-suspension cultures derived from transgenic Nicotiana benthamiana leaves expressing wild-type or chimeric Bamboo mosaic virus (BaMV) transgene carry viral protein 1 (VP1) epitope of foot and mouth disease virus (FMDV) at the N-terminus of coat protein (BVP1). The expression of BVP1 protein was detected in cell-suspension cultures by Western blot and ELISA. Subsequently, a high level of chimeric BaMV particles (CVPs) were purified from BdT38 and BdT19 transgenic cell-suspension cultures co-expressing silencing suppressor protein P38 and P19, respectively (~ 1.5 or 2.1 mg/20 g of suspended fresh weight biomass) when compared with transgenic line BVP1-16-7, in which this silencing suppressor protein expression is absent. Furthermore, immunoelectron microscope analysis revealed the VP1 epitope expression on the surface of CVPs. Subsequently, guinea pigs vaccinated with these CVPs particles have generated neutralizing antibodies. We have developed a prototype protocol to simplify the purification procedure using size-exclusion chromatography to replace the traditional centrifugation based virus purification. Our results showed a remarkable recovery of CVPs from cell-suspension cultures. VP1 epitopes were displayed on the surface of CVPs. Our studies provide insight to generate immunopeptide vaccines in a cost effective manner, as well as streamline the methods for a large-scale production using natural plant-based milieu that offers an alternative and efficient vaccines source.
URI: http://hdl.handle.net/11455/90038
文章公開時間: 10000-01-01
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