奈瑟氏菌表面抗原基因轉殖至水稻之研究

Studies on the Surface Antigenic Gene of Neisseria gonorrhoeae Transferred into Rice (Oryza sativa L.)

劉芯瑄(Hsin-Hsuan Liu) ； 曾夢蛟(Menq-Jiau Tseng) ； 楊秋英(Chiou-Ying Yang) ； 郭純德(Chun-Teh Kuo) ； 尤進欽(Jinn-Chin Yiu)

《宜蘭大學生物資源學刊》 4卷1期 (2008/06) Pp. 47-57

https://doi.org/10.6175/job.2008.41.15

摘要

奈瑟氏淋病雙球菌（Neisseria gonorrhoeae）是常見的傳染疾病，且仍是全世界主要的問題。成人通常經由性接觸傳染淋菌。由於抗生素濫用，造成抗藥性淋病增加。因此，利用疫苗抗原選殖至可食用轉殖植物使成為口服性疫苗，將是一個新興的運送系統。本試驗將分離自奈瑟氏菌表面抗原基因（Ag473）構築以肌動蛋白（actin）啟動子驅動之植物轉殖載體，並利用農桿菌法轉殖至水稻真核表達系統中表現。本研究的目的為轉殖Ag473至水稻系統的建立，及探討目標蛋白於水稻品種中之表現情形。本實驗之轉殖材料是以水稻（`台稉8號´）子葉盤誘導的癒傷組織。將再生的癒傷組織藉由農桿菌轉移後以kanamycin初步篩選。轉型存活之再生植株萃取其RNA進行RT-PCR分析，確定插入水稻染色體的Ag473基因正常轉錄成mRNA。再利用Ag473抗體蛋白進行葉片與種子的西方墨點分析，確認轉殖水稻正確的合成與累積目標蛋白。Ag473蛋白之表現量以再生水稻種子進行分析，約佔總可溶性蛋白的0.36～0.41%。子代分析上，轉殖再生水稻T1後裔進行RT-PCR分析，均可檢測到Ag473 RNA之訊號，顯示目標基因高度穩定遺傳至後代。因此，本實驗建議水稻可做為奈瑟氏菌表面抗原（Ag473）基因表達的理想候選者。

關鍵字

奈瑟氏菌 ； 表面抗原 ； 水稻 ； 食用疫苗

並列摘要

Neisseria gonorrhoeae is a common infectious disease of humans and has remained a major problem world-wide. N. gonorrhoeae commonly transmits in adults through sex contact. Antibiotic-resistant N. gonorrhoeae highly emerges through the abuse of antibiotic. The cloning of antigen into edible transgenic plants is a promising delivery system for oral vaccines. The plant nuclear expression vector with overproduction of Ag473 gene driven by actin promoter was constructed. Agrobacterium tumefaciens containing the Ag473 expression constructs was used to transform rice. The objective of this study is to establish Agrobacterium transformation of Ag473 rice, and to evaluate the expression of targeted protein in rice. The scutellar-derived embryogenic calli of original rice of variety (`TK8´) were used in this study. Regenerated plant transformed with Ag473 gene via Agrobacterium were selected using kanamycin. Transcription RNA of Ag473 gene was examined by reverse transcription RT-PCR. Western blot analysis using antiserum against Ag473 was carried out to determine the expression of protein in transgenic rice cells. The Ag473 protein specifically expressed and accumulated in seeds was at a level of 0.36~0.41% of the total seed protein in transgenic plants. Stable integration and expressing RNA of the Ag473 gene in T1 progeny were confirmed by RT-PCR. These results suggested that the transgenic rice cells with expression of Ag473 protein were the ideal candidates.

並列關鍵字

Neisseria gonorrhoeae ； surface antigen ； rice ； edible vaccine