乙肝病毒表面抗原基因植物表达载体的构建及其在烟草和番茄中的表达
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摘要
乙型肝炎是一种严重危害人类健康的全球性疾病,接种乙肝疫苗是目前预防乙肝病毒(HBV)感染的主要措施。乙肝病毒表面抗原(HBSAg)为HBV包膜蛋白的组分,不具感染性,是乙肝疫苗的有效成分。现行乙肝疫苗主要通过酵母表达系统得到,其生产工序复杂、需要专门的纯化设备和相应的技术人员,成本较高,从而限制了乙肝疫苗的广泛应用,而利用植物生产乙肝疫苗可有效弥补这一不足。因此,本实验构建了含乙肝病毒表面抗原基因的植物表达载体,并将其分别在烟草和番茄中成功进行了表达。
本实验首先构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,具CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于根癌农杆菌的转化;其次将表达质粒导入农杆菌,用于对烟草外植体的遗传转化,获得的抗性植株经检测,结果表明乙肝病毒表面抗原基因在烟草中得到表达;最后,将HBSAg基因用于番茄的遗传转化,其间建立了番茄再生体系以及番茄遗传转化体系,获得的抗性植株经检测,证明HBSAg基因在番茄植株中也同样获得了表达,这为利用转基因番茄生产口服疫苗提供了一定的理论指导和实验基础。
Hepatitis B is a contagious disease that damages the people's health all over the world seriously.It is a main method for most people to inoculate the vaccine against the hepatitis B virus.The hepatitis B surface antigen is an ingredient of HBV envelop protein, which is not contagious and is the effective composition of the hepatitis B vaccine.The hepatitis B vaccine can not be used widely because nowadays the production of hepatitis B vaccine is carried out via yeast expression system,its procedure is complicated,it needs the special equipment for purification and some technicians,therefore its cost is very high.It can be solved this problem for us to utilize transgenic plants to produce the oral hepatitis B vaccine.In this paper,the plant expression vector containing hepatitis B surface antigen gene has been constructed and transformation of HBSAg gene into tobacco and tomato plants has been carried out in order to obtaine transgenic plants.
The experiment was performed and the results were shown as follows:First of all, the plant expression vector pBRSAg was constructed,which contained all elements for plant expression and was suitable for transformation via Agrobacteria,such as CaMV35S promoter,both left and right border sequence for T-DNA in Agrobacteria,plant reporter gene gus,plant screening marker gene hpt.Second,the plant expression vector pBRSAg was introduced into Agrobacteria.Third,it was carried out to tansform the HBSAg gene into tobacco plants,transgenic tobacco plants were obtained,the detection was performed and the result indicated that the HBSAg gene was expressed in transgenic tobacco plants. Fourth,the systems of the tomato regeneration and transformation were established and the introduction of the HBSAg gene into tomato plants was performed,the result was shown that the HBSAg gene was also expressed in transgenic tomato plants.These results might provide the theoretical and practical bases for studying the production of oral hepatitis B vaccine ultilizing transgenic tomato plants.
本实验首先构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,具CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于根癌农杆菌的转化;其次将表达质粒导入农杆菌,用于对烟草外植体的遗传转化,获得的抗性植株经检测,结果表明乙肝病毒表面抗原基因在烟草中得到表达;最后,将HBSAg基因用于番茄的遗传转化,其间建立了番茄再生体系以及番茄遗传转化体系,获得的抗性植株经检测,证明HBSAg基因在番茄植株中也同样获得了表达,这为利用转基因番茄生产口服疫苗提供了一定的理论指导和实验基础。
Hepatitis B is a contagious disease that damages the people's health all over the world seriously.It is a main method for most people to inoculate the vaccine against the hepatitis B virus.The hepatitis B surface antigen is an ingredient of HBV envelop protein, which is not contagious and is the effective composition of the hepatitis B vaccine.The hepatitis B vaccine can not be used widely because nowadays the production of hepatitis B vaccine is carried out via yeast expression system,its procedure is complicated,it needs the special equipment for purification and some technicians,therefore its cost is very high.It can be solved this problem for us to utilize transgenic plants to produce the oral hepatitis B vaccine.In this paper,the plant expression vector containing hepatitis B surface antigen gene has been constructed and transformation of HBSAg gene into tobacco and tomato plants has been carried out in order to obtaine transgenic plants.
The experiment was performed and the results were shown as follows:First of all, the plant expression vector pBRSAg was constructed,which contained all elements for plant expression and was suitable for transformation via Agrobacteria,such as CaMV35S promoter,both left and right border sequence for T-DNA in Agrobacteria,plant reporter gene gus,plant screening marker gene hpt.Second,the plant expression vector pBRSAg was introduced into Agrobacteria.Third,it was carried out to tansform the HBSAg gene into tobacco plants,transgenic tobacco plants were obtained,the detection was performed and the result indicated that the HBSAg gene was expressed in transgenic tobacco plants. Fourth,the systems of the tomato regeneration and transformation were established and the introduction of the HBSAg gene into tomato plants was performed,the result was shown that the HBSAg gene was also expressed in transgenic tomato plants.These results might provide the theoretical and practical bases for studying the production of oral hepatitis B vaccine ultilizing transgenic tomato plants.
引文
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