【作者】 刘科；

【导师】 尉亚辉； 张洪霞；

【作者基本信息】 西北大学 ， 植物学， 2004， 硕士

【摘要】 乙型肝炎是由乙型肝炎病毒(hepatitis B virus，HBV)引起的一种世界性疾病，据统计全球HBV慢性携带者的总数超过2.8亿。中国是乙型肝炎的高流行区，有20％的人感染过。预防乙肝最有效的途径是接种疫苗，然而目前使用的疫苗具有种种限制性因素，而利用转基因植物生产疫苗有望克服传统疫苗所具有的种种缺点。 本试验利用从乙肝患者血清中提取的乙肝病毒基因组DNA为模板，依据Genbank登录号为AY040804的序列设计含有Kozak序列的引物，扩增出乙肝表面抗原s蛋白基因(HBsAg)，经BLAST比对，确认其中有1个碱基发生突变，经分析很可能为一种新的乙肝病毒毒株，现已提交GenBank进行处理。 利用克隆得到的HBsAg基因与二元植物载体pBI121成功地构建出含有CaMV35s启动子、nos中止子、Kan植物筛选标记及HBsAg基因的植物表达载体pBI121-HBsAg。 并通过冻融法将此质粒转化入土壤农杆LBA4404，侵染烟草叶片，目前长势良好，正处在筛选当中。 通过以上实验，将建立起适用于本实验室的烟草、番茄高效遗传转化体系；并利用CaMV35s高效启动子启动HBsAg基因在烟草中表达，获得具有免疫活性物质的再生植株，为利用转基因植物生产食用疫苗提供一定的试验基础。更多还原

【Abstract】 Hepatitis B is a world wide infectious disease which is induced by HBV (Hepatitis B virus). There are 280 million patients with HBV in the world. China is a high infectious zone with 20% people infected by HBV. HBV vaccination is the best way to prevent the infection of HBV, but the vaccine used now has many disadvantages. Now producing HBV vaccine with transgenic plant can overcome the traditional vaccine’s shortcoming.In this experiment, we isolated HBV genome DNA from the serum that was obtained from the patient who infected HBV, and designed a pair of primers with Kozak consensus sequence based on the sequence AY040804 obtained in Genbank. From which, we amplified HBsAg gene that coding hepatitis B virus surface antigen S protein. By BLAST, we confirmed 1 base in this sequence was mutated. Perhaps it’s a kind of new virus strain, and the sequence was submitted to Genbank, it is processing now.Moreover, we constructed a new plant expression vector-pBI121-HBsAg with HBsAg gene and pBI121 binary vector. pBI121-HBsAg contains CaMV35s promoter, nos terminator, Kan marker gene and HBsAg gene, and this plasmid was transformed to agrobacterium LBA4404 by freeze and thaw method, and then the tobacco leaf disc was transformed by LBA4404.Through this experiment, we want to establish a high efficiency transform system for tomato and tobacco; use CaMV35s high efficiency promoter to promote expression of HBsAg in tobacco, obtain regeneration plants contain specific protein with immunocompetence, and build a foundation for the further research of transgenic plant based vaccine.更多还原