神經氨酸酶基因轉殖至番茄之研究

A Study on the Transfer of Neuraminidase Gene into Tomato

流行性感冒是由流感病毒（Influenza virus）所引起的急性呼吸道感染疾病，幾乎每年都會流行，有時有高發病率及致死性的全球大流行。為了供應全球對流感疫苗的需求，有發展一個新的疫苗製造策略的必要。本研究之目的係將構築於植物載體之神經氨酸酶（neuraminidase，NA）基因，以農桿菌轉殖法轉移至番茄，並探討它在轉殖番茄中之表現情形。使用之轉殖材料為‘金珠’番茄。NA基因經由農桿菌轉殖法轉移至番茄子葉後，以100 mg．L^(-1)kanamycin進行篩選，並且誘導再生。轉殖植株之再生率約為7.5%。由轉殖成功之再生植株萃取其DNA進行PCR分析，檢驗18株中有17株具有反應條帶，證實NA基因插入番茄基因組中。17株轉殖株中有13株的RT-PCR分析呈正反應，顯示插入番茄基因組的NA基因可正常轉錄。西方墨點分析中發現，NA蛋白於葉片及果實中的平均表現量分別佔總可溶性蛋白的0.067%和0.032%；以葉片之表現量較高。本試驗結果顯示利用番茄作為表現人類流行性感冒病毒表面抗原基因之可行性，且目標基因可成功於轉殖番茄中累積神經氨酸酶蛋白。 Influenza is an infectious disease caused by influenza viruses which lead to common symptoms of acute respiratory infections. Influenza epidemic occurs almost every year and global pandemics with high morbidity and mortality occur less frequently. Alternative strategies may be considered in order to supply global demands for influenza vaccines. In this study, a nuraminidase gene (NA) was constructed in a plant expression vector, introduced into tomato genome through Agrobacterium-mediated transformation and its expression in the transgenic tomatoes explored. The cotyledons of ‘Golden Gem’ tomato as explants was cocultivated with Agrobacterium and screened in a regeneration medium containing 100 mg．L^-1 of kanamycin. The regeneration percentage was 7.5%. To identify the presence of the NA gene in tomatoes, genomic DNA was isolated from putative transgenic plantlets. By using PCR, 17 out of the 18 regenerated plantlets were identified to contain the introduced NA fragment, suggesting a successful integration into the genome of transgenic tomato plants. The expression of the NA gene in these 17 independent transgenic plants was examined by using reverse transcription PCR (RT-PCR). The results revealed that NA transcript could be identified in 13 transgenic plants. In the western blot analysis, NA protein was detected in leaves and fruits at the average expression levels of 0.067% and 0.032% of the total soluble protein, respectively. These results demonstrate the feasibility of producing human influenza virus surface antigen in transgenic tomatoes.