霍乱肠毒素B亚单位在转基因番茄果实中特异性表达及其免疫原性的研究  

Transgenic Tomato Plant Expressing Cholera Toxin B Protein Specifically in Fruit as Edible Vaccine

作　　者：江晓玲[1] 贺竹梅[2] 陈清[1] 彭志强[1] 祁瑜[2] 俞守义[1] 

机构地区：[1]第一军医大学热带军队卫生学系流行病学教研室,广州510515 [2]中山大学生命科学院生物工程中心,广州510275

出　　处：《中国农业科学》2004年第8期1188-1192,共5页Scientia Agricultura Sinica

基　　金：广东省自然科学基金重点资助项目(013075)

摘　　要：利用番茄果实特异性启动子-E8启动子,将霍乱肠毒素B亚单位基因(ctb)用根癌农杆菌浸润法转入番茄植株,并使其在果实中特异表达,然后对试验小鼠进行口服免疫,研究转基因番茄果实的免疫原性。结果表明,ctb基因在14株番茄植物基因组中被证实有整合,并在其中2株的番茄果实中有特异性表达,最高表达量分别为455和385 ng·g-1FW,口服免疫后的小鼠血液和肠道粘膜中均检测到抗CTB抗体。表明获得了在番茄果实中特异、高效表达霍乱肠毒素B亚单位基因(ctb)的植物口服疫苗候选植株。To make cholera toxin B gene(ctb) expressed in ripening tomato fruit under control of fruit-specific E8 promoter and detect its immunity as an edible vaccine by oral immunization of mice, plasmid pJES 1 containing ctb gene with E8 promoter was induced into Agrobacterium tumefaciens strain LBA4404 and used to transform cotyledons of tomato. Kanamycin-resistant shoot regenerants were selected, elongated, rooted and transferred for fruiting in greenhouse. Transgenic plants were confirmed by PCR and Southern blot analysis. Proteins were extracted from leaf, stem, and fruit tissue of transgenic plants and detected by ELISA and Western blot. Serum and mucosal samples drawn from oral immunized and preimmunized mice were analyzed by ELISA. 14 out of 26 kanamycin-resistant shoot regenerants were confirmed to be transgenic by PCR and Southern blot. ELISA analysis demonstrated that recombinant CTB antigen was expressed in fruits of transgenic plants 9 th and 16 th respectively, and the highest amount of CTB antigen reached 455 and 385 ng·g-1FW in ripening tomato fruits. Oral immunization of mice with ripening fruits of plant 9 th led to the induction of both serum and mucosal CTB specific antibodies. Transgenic tomato plant expressing cholera toxin B protein specifically in fruit as edible vaccine was then obtained successfully.

关 键 词：霍乱肠毒素 B亚单位 转基因番茄 果实 特异性表达 免疫原性 

分 类 号：Q789[生物学—分子生物学]