【摘要】 目的提高乙型肝炎病毒表面抗原(HBsAg)在人参愈伤组织细胞中的表达。方法构建一种植物细胞表达载体pBIBeo,该载体携带CaMV 35S双增强启动子和TMV(U1株)翻译增强子。用pBIBeo转化的农杆菌LBA4404与人参愈伤组织细胞共培养,经G418筛选获得新生抗性细胞团PBIBeo,连续继代培养选育出用于植物源口服乙肝疫苗生产的高产细胞株。提取基因组DNA和mRNA,进行PCR和RT-PCR鉴定,Western blot鉴定HBsAg的特异性,电镜下观察纯化抗原颗粒大小,并以ELISA检测HBsAg的表达水平。结果转化的人参细胞基因组DNA进行PCR反应,得到约700 bp的启动子基因片段。提取mRNA进行RT-PCR反应,得到约700 bp的HBsAg基因片段。Western blot分析可见相对分子质量为24 000的特异性条带。亲和层析纯化细胞表达的HBsAg抗原,电镜观察可见平均直径为32 nm的颗粒。ELISA检测结果表明pBIBeo转化细胞株HBsAg表达量比pBIBSa转化细胞株提高1~2倍。结论人参细胞基因组已经整合了目的基因并稳定高效表达。更多还原

【Abstract】 Objective To increase the expression level of HBsAg in ginseng callus cells. Methods Construct a plant expression vector pBIBeo containing CaMV 35S promoter with duplicated enhancer and translation enhancer of tobacco mosaic virus(TMV)strain U1 and transform to Agrobacterium tumefaciens(strain LBA4404).Co-culture the transformed strain LBA4404 with ginseng callus cells and screen the nascent resistant cell aggregates PBIBeo with G418 for continuous secondary culture to screen cell strain used for production of plant-derived oral hepatits B vaccine.Extract genomic DNA and mRNA and identify by PCR and RT-PCR respectively.Determine the expression level of HBsAg by ELISA and identify the specificity by Western blot.Observe the purified HBsAg particles by electron microscopy. Results The promoter gene fragment at a length of about 700 bp was amplified from the genomic DNA of ginseng cells transformed with recombinant plasmid pBIBeo by PCR.The HBsAg gene fragment at a length of about 700 bp was amplified from mRNA of the transformed ginseng cells by RT-PCR.Western blot showed a specific band with relative molecular mass of 24 000.The mean particle size of purified HBsAg by affinity chromatography was 32 nm.ELISA proved that the expression level of HBsAg in pBIBeo-transformed ginseng cells was 1～2 times higher than that in pBIBSa-transformed ginseng cells. Conclusion The target gene was integrated to the genome of ginseng cells and expressed stably and highly.更多还原