幽门螺旋杆菌相关蛋白CagA、UreB基因与CTB融合基因在番茄中的遗传转化

发布时间：2018-06-30 03:17

本文选题：幽门螺旋杆菌 + CagA基因　；参考：《浙江大学》2006年硕士论文

【摘要】： 幽门螺杆菌是慢性胃炎、消化性溃疡的病原体，也与胃腺癌及黏膜相关性淋巴瘤的发生密切相关。各国人群中Hp感染率约为50％，发展中国家超过70％。世界卫生组织国际癌症研究机构已将该菌纳入第一致癌原。CTB是一个有效的载体分子，抗原分子与CTB结合后，，与肠相关淋巴组织(Gut—associated lymphoid tissues，GALT)细胞表面的神经节苷脂GM_1具有较强的亲和力，这就更利于抗原的传递和免疫识别，引起口服耐受。番茄是基因工程中最为广泛使用的模式植物之一并且番茄的果实可以直接实用是研制植物口服疫苗的理想植物体。本研究构建了含有融合基因CTB—UreB的果实特异启动子植物表达载体，并将已构建好的含有融合基因CTB—CagA、CTB—UreB的植物表达载体，采用农杆菌介导的叶盘转化法，将融合基因转入番茄中，为利用生物反应器研制口服型植物疫苗奠定了先行基础。研究结果表明如下： ①利用农杆菌介导的遗传转化系统将融合基因转入番茄基因组中，经过共培养和多次抗性筛选、诱导不定芽的分化和生根，得到了具有抗性的再生植株。再生植株的抗性芽分化率达40～60％。 ②PCR和RT—PCR验证目的基因已经整合到了番茄的基因组中，PCR阳性率＞70％。 ③筛选出来的阳性植株继续培养待果实成熟后取种子(F_1代)，将F_1代种子播种培养，经过检测所播种的第二代植株中阳性率＞90％。 ④构建了番茄果实特异启动子表达载体，经酶切检验正确后可用于转化番茄外植体。
[Abstract]:Helicobacter pylori is the pathogen of chronic gastritis, peptic ulcer and gastric adenocarcinoma and mucosal associated lymphoma. The infection rate of HP is about 50% in the population of different countries and more than 70% in the developing countries. The World Health Organization's International Agency for Cancer Research has incorporated the bacterium into the first carcinogen. CTB is an effective vector molecule, and the antigen molecule binds to CTB. GM-1 has strong affinity with GM1 on the surface of Gut-associated lymphoid tissue (GALT) cells, which is more favorable for antigen transmission and immunological recognition, resulting in oral tolerance. Tomato is one of the most widely used model plants in genetic engineering. In this study, the fruit specific promoter plant expression vector containing CTB-UreB was constructed, and the plant expression vector containing CTB-CagAN-CTB-UreB was constructed by Agrobacterium tumefaciens-mediated leaf disk transformation. The transfer of the fusion gene into tomato lays the foundation for the development of oral plant vaccine by bioreactor. The results are as follows: 1 the fusion gene was transferred into tomato genome by Agrobacterium tumefaciens mediated genetic transformation system. The resistant regenerated plants were obtained by inducing the differentiation and rooting of adventitious buds. The resistant bud differentiation rate of regenerated plants reached 400.2The PCR and RT-PCR confirmed that the target gene had been integrated into tomato genome and the positive rate of PCR was > 70. (3) the selected positive plants continued to be cultured after fruit ripening, and the seeds were sowed and cultured in the first generation. The expression vector of tomato fruit specific promoter was constructed by detecting the positive rate of the second generation plantlets sown with a positive rate of > 90.4, which can be used to transform tomato explants by enzyme digestion test.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q943.2;R392

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