- 學位論文
植物生產外源蛋白質之研究
Studies on Expression of Heterologous Proteins in Plants
摘要
植物作為生產次單位口服疫苗之生物反應器，已於不同植物材料間發展出多種生產策略，突破既有之微生物或動物表達醫藥用蛋白質模式，能快速獲得穩定及高品質之醫藥用蛋白質。本論文以植物病毒載體 (viral-based vectors) 暫時性感染、穩定性核基因轉殖 (nuclear transformation) 及葉綠體基因轉殖 (chloroplast transformation) 等三種策略，於不同植物表達重組豬生殖和呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus, PRRSV) 之次單位疫苗，包括封套醣蛋白 (envelope glycoprotein 5, GP5)、膜蛋白 (membrane protein, M) 與具佐劑功能之大腸桿菌 (Escherichia coli) 熱不穩定毒素B次單位 (heat-labile toxin B subunit, LT-B)。植物病毒載體選用蘭花之齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV)，首先分析ORSV 鞘蛋白 (coat protein, CP) 次基因組核醣核酸 (subgenomic RNA, sgRNA) 啟動子活性，顯示CP sgRNA啟動子位於轉譯起始點-290至+18核苷酸 (nucleotide, nt) 間，啟動子短至8 bp即具轉錄活性，-24至+18 nt具有最高的表達量，推測為ORSV CP啟動子核心區域。應用農桿菌針筒注射滲入法 (syringe agroinfiltration) 及真空抽氣滲入法 (vacuum agroinfiltration) 於蝴蝶蘭 (Phalaenopsis) 花瓣、邊沁菸草 (Nicotiana benthamiana) 及其他植物葉片中進行ORSV表達載體暫時性表達，顯示於ORSV cDNA 3’端構築含D型肝炎 (hepatitis delta virus, HDV) 或錘頭型 (hammerhead) 核醣核酸酵素 (ribozyme) 序列，皆能提升GP5蛋白質表現量，佔總可溶性蛋白 (total soluble protein, TSP) 0.043%~0.063%。觀察穩定性核基因轉殖之香蕉植株，生長及外表性狀正常，果實可自然後熟並產生香氣，其表達GP5蛋白質含量約佔0.074% TSP。進行葉綠體轉殖則使用披衣藻 (Chlamydomonas reinhardtii) 表達次單位疫苗，根據披衣藻葉綠體密碼子使用偏性 (codon usage)，構築得優化LT-B/ORF6/ORF5融合基因表達質體，以基因槍轉殖法轉殖於披衣藻葉綠體中，經抗生素篩選取得轉殖系後，鑑定蛋白質顯示次單位融合蛋白質成功於轉殖披衣藻中表達，其蛋白質表達量約佔0.065% TSP。
並列摘要
Application of plants as a bioreactor for production of subunit oral vaccine has been successfully developed in different plant species to obtain stable and high-quality pharmaceutical proteins. In this study, fused of envelope glycoprotein (GP5), membrane protein (M) of porcine reproductive and respiratory syndrome virus (PRRSV) and heat-labile toxin B subunit (LT-B) of Escherichia coli were expressed in different plants by three strategies: transient expression by viral-based vectors, stable nuclear transformation, and chloroplast transformation. Deletion analysis of Odontoglossum ringspot virus (ORSV) coat protein subgenomic RNA (sgRNA) promoter activity suggested that the minimal CP sgRNA promoter is located between -290 to +18 relative to translation start site (TSS). As short as 8 bp upstream to TSS was sufficient to direct the expression of reporter genes. The -24~+18 around TSS of CP was with the highest expression level and could be the core promoter. ORSV-based viral vectors and revised versions were transformed into petals of Phalaenopsis and leaves of Nicotiana benthamiana by syringe agroinfiltration or vacuum agroinfiltration for transient expression. Addition of hepatitis delta virus (HDV) or hammerhead ribozyme sequences increased at the 3’-end of ORSV cDNA vector containing the expression of GP5 protein from 0.43% to 0.063% of total soluble protein (TSP). Growth and phenotype of nuclear transgenic banana were normal and protein contents of GP5 expressed in transgenic banana fruits about 0.074% of TSP. For chloroplast tramsformation, codons in LT-B/ORF6/ORF5 fused gene were optimized according to the codon usage of Chlamydomonas reinhardtii chloroplast gene bias and transferred into the chloroplast of C. reinhardtii by particle bombardment. The GP5 protein expressed in transgenic C. reinhardtii lines were sequenced and quantified as about 0.065% of TSP after selection by antibiotics.