乙肝病毒表面抗原基因在人参细胞中的高效表达

发布时间：2018-01-16 21:24

本文关键词：乙肝病毒表面抗原基因在人参细胞中的高效表达　出处：《吉林大学》2007年博士论文　论文类型：学位论文

【摘要】： 我们在前期工作中建立了人参愈伤组织细胞表达乙肝病毒表面抗原(HBsAg)的植物表达系统,并就提高表达进行了培养条件的摸索。为了进一步提高HBsAg的表达,我们从表达载体的改造上做了一些探索,对调控HBsAg基因表达的启动子以及翻译增强子进行了改造,构建了十二种不同的植物细胞表达载体pBI/EP、pBI/2EP、pBI/3EP、pBI/4EP、pBI/5EP、pBI/6EP、pBI/7EP、pBI/8EP、pBIBSa(pBI35S)、pBI/2EPomega、pBI/35Somega和pBI/35SRomega,用这些载体分别转化农杆菌LBA4404,再与人参愈伤组织细胞共培养,经G418筛选获得新生抗性细胞,从而将HBsAg基因以及调控基因整合到人参细胞基因组中,经过连续继代培养选育出一株高产细胞系PBIo。 PBIo是由质粒pBI/35Somega转化的人参细胞选育而来,携带花椰菜花叶病毒CaMV 35S启动子以及与之串联的烟草花叶病毒TMV(U1株)翻译增强子omega序列。通过对表达载体的改造使HBsAg基因表达有显著提高。在实验中还发现,omega反义RNA序列也具有较强的提高翻译效率的功能。
[Abstract]:A plant expression system for the expression of hepatitis B virus surface antigen (HBsAg) in callus cells of Panax ginseng was established in our earlier work. In order to further improve the expression of HBsAg, we made some exploration on the transformation of expression vector. The promoter and the translation enhancer regulating the expression of HBsAg gene were modified and 12 different plant cell expression vectors pBI / EPP / 2 EPBI / 3 EP were constructed. PBI / 4EPB / 5EPBI / 6EPBI / 7EPBI / 8EPBI / 8EPBI35Sv PBI / 2EPomega; PBI / PBI / 5EPBI / 5EPBI / 6EPB / 7EPBI / 8EPBI / 8EPBI35Sv / PBI / 2EPomega. PBI/35Somega and pBI / 35 SRomega were transformed into Agrobacterium tumefaciens LBA4404 respectively and co-cultured with ginseng callus cells. New resistant cells were obtained by G418 screening, and then the HBsAg gene and regulatory gene were integrated into the genome of ginseng cells, and a high-yielding cell line PBIoO was selected after successive subculture. PBIo was selected from ginseng cells transformed by plasmid pBI/35Somega. Carrying CaMV 35s promoter of cauliflower mosaic virus and TMV(U1 strain of tobacco mosaic virus (TMV). Translation enhancer omega sequence. Through the modification of expression vector, the expression of HBsAg gene was significantly improved. It was also found in the experiment. Omega antisense RNA sequences can also improve translation efficiency.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392;Q943.2

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