【作者】 马英；

【导师】 林顺权； 吕柳新；

【作者基本信息】 福建农林大学 ， 果树学， 2002， 博士

【摘要】 本研究以番茄子叶和下胚轴为材料，以Gus瞬时表达效率为指标，系统研究了农杆菌介导的番茄遗传转化的影响因素，在此基础上建立并优化了番茄“羞女”品种的遗传转化体系；构建乙型肝炎病毒表面抗原基因（HBsAg）、戊型肝炎病毒ORF₂基因片段NE₂（HEV-NE₂）的植物表达载体，利用已建立的转化体系将其导入番茄，获得了经PCR和Southern杂交证实的转基因植株。ELISA检测结果显示在番茄中表达的目的蛋白具有较好的免疫活性；动物试验结果初步表明，用转基因番茄表达的HBsAg口服或肌注初免过的小鼠，均有不同程度的加强免疫效果。展示了用番茄研制乙肝、戊肝口服疫苗的美好前景，为推动转基因植物口服疫苗走向实用化提供实验基础和理论指导。 1．植物表达载体构建：利用含GUS和去GUS的中间载体pCAMBIA1301和pCAMBIA1300构建了乙肝病毒表面抗原（HBsAg）、戊肝病毒ORF₂基因片段NE₂的植物表达载体p1301HBs、p1300HBs、p1301NE₂和p1300NE₂。其中含CaMV35s启动子、nos终止子、Ω增强子、Kan抗性筛选标记Kan^r和潮霉素磷酸转移酶基因（HPT）。经酶切鉴定后直接法导入农杆菌菌株LBA4404、EHA105、AGL1。 2．农杆菌介导的番茄遗传转化体系的建立和优化：以含质粒p1301HBs（含Gus基因）的根癌农杆菌侵染番茄外植体，以Gus瞬时表达效率为指标，系统研究了农杆菌菌株、外植体年龄、预培养时间、共培养时间、培养条件、筛选条件等对转化效率的影响并在此基础上建立、优化了番茄“羞女”品种的遗传转化体系。 福建农林大学博士学位论文 中文摘要 即：EHA 105菌株；外植体苗龄gd暗光下预培养3d，或苗龄12d 暗光下预培养Zd：农杆菌侵染后暗光下共培养3小筛选剂潮霉素 （Hrr）浓度为20mm／L，杀菌剂前期用梭芋青霉素（Carb），后期 用头抱霉素（Cef），浓度初期 500mg／L，以后逐渐降低。 将该体系运用于去 GUS标记的 PI 300HBS和戊肝病毒基因的 转化，得到了较好的结果，说明该体系是可靠、可重复、有效的。 3．转基因苗的鉴定及目的蛋白的定量：抗性苗总DNA经 PCR、Southern杂交证实目的基因己整合到番茄基因组中，ELISA 检测结果证明在番茄中正确表达了具有免疫活性的乙肝病毒表面 抗原蛋白门BSAg人戊肝病毒ORFZ基因片段NEZ(HEV－NEZ人 根据ELISA定量结果，HBSAg在番茄中最大表达量约为240刁00 us／s，约占总可溶性蛋白的0．003％，而eV－NEZ的表达量较低。 目前己扩繁转HBSAg的转基因苗40多株，已结番茄100多个，保 存有转HEV十 的转基因苗7株，其中有一株己结两个果实。 4．小鼠免疫试验：以0．sllg／g亚免疫剂量初兔的小鼠，4 周后用转HBSAg的转基因组织饲喂，至第8周抗体由饲喂前的 0．4上升到互．0以上；用浓缩、纯化过的植物蛋白加强免疫初免 过的小鼠，也发现有较强的特异性抗体上升。说明用番茄表达的 重组HBSAg具有免疫原性。更多还原

【Abstract】 By using cotyledons and hypocotyls of tomatos as transformatic explants, the factors affecting gene transfer mediated by Agrobacterium tumefaciens were systematically examined according to Gus expressing efficiency, and the optimized genetic transformation system of tomato was established. The plant binary expression vector p!301HBs, pBOOHBs carrying hepatitis B virus surface antigen(HBsAg) gene, p!301NE2, p!300NE2 carrying NE2 gene, a fragment of ORF2 region of hepatitis E virus (HEV-NE2), were constructed and introduced into tomato, and hygromycin-resistant plantlets were obtained. The results of PCR and Southern blotting of tomato genomic DNA demonstrated that the target gene was integrated into the genome of tomato plants. The examination of ELISA suggested the HBsAg gene has expressed correctly in transgenic tomato plants. The immunization experiment indicated that the HBsAg-transgenic plant derived vaccine can produce booster immune responses in Balb/c mice primed with commercial vaccine. All these demonstrated the perfect perspective of plant-derived edible vaccine and provided experimental basis and guiding theory for the practicality of plant-derived edible vaccine.1.Construction of plant binary expression vector: The HBsAg gene linked with CaMV35S promoter, nos terminal and Q enhancer was inserted into the mediate expression vectors pCAMBIA1301 (containing hygromycin- resistant gene, kanamycin-resistant gene, Gus gene) and pCAMB!A1300 (containing Hyg- resistant gene, Kan-resistant gene, no Gus gene), respectively, to form the reconstructed plant binary expression plasmids p!301HBs and p!300HBs. In the same way, the plant binary expression plasmids p!301NE2, p!300NE2 carrying HEV-NE2 gene were constructed. Restriction endonuclease analysis indicated that these plasmids were directly introduced into strains LBA4404, EHA105, AGL1.2. Genetic transformation system establishment: Genetic transformation conditions were examined by Gus expression, and an efficient genetic transformation system was developed. That is: 9-days-old explants pre-cultured for 3 days in darkness or 12-days-old explants pre-cultured for 2 days in darkness were co-cultured with EH A105 for 3 days in darkness, then selected by 20mg/g hygromycin. 500mg/g carbenicillin(carb) which will be replaced by cefotaxine(cef) later was used as inhibitor of the Agrobacterium tumefaciens, both of which will be reduced gradually.Using the above-mentioned transformation system, the p!300HBs without Gus gene, and the HEV-NE2 gene were introduced into tomato, many hyg-resistent plantlets were acquired too. This demonstrated that the established transformation system is reliable, repeatable, thus efficient.3. Distinguishing the hyg-resistant plantlets and determining expression amount of target protein: The integration of foreign DNA into transgenic tomato was confirmed by hygromycin resistance, Gus assay, PCR and Southern blotting. The immunoacticity of recombinant HBsAg, HEV-NE2 was shown by ELISA. The amount of HBsAg in transgenic tomato is about 240-300ng/g fresh weight, and 0.003 percent of total soluble protein, whereas the amount of HEV-NE2 is very low. Up to now, we have obtained about 40 HBsAg-transgenic plants bearing more than 100 tomato fruits, 7 NE2~transgenic plants, one of which has born 2 fruits.4. Immunogenicity of transgenic plant-derived HBsAg: Mice primed with injection of a sub-immunogenic dose(0.5 u g/g) of commercial vaccine, were fed with transgenic tissue or injected with purified extraction. Four weeks later, the mice boosted special serum antibody of HBsAg.更多还原