摘要
为获得表达乙肝病毒表面抗原(HBsAg)的人参细胞系,构建携带HBsAg基因的植物细胞表达载体pBIBSa,采用以农杆菌LBA4404感染的方法,经G418筛选后得到13株具有抗性的人参细胞。提取基因组DNA进行PCR反应,其中8株得到约700bp的HBsAg基因片段;提取mRNA进行RT-PCR反应,其中6株得到约700bp的HBsAg基因片段;用ELISA方法检测HBsAg,6株均为阳性。每克人参细胞中最高含HBsAg184ng,占细胞可溶性蛋白的0.009%。免疫组化切片染色显示HBsAg主要分布在细胞膜上,少量位于细胞核中。这些结果表明人参细胞整合了HBsAg基因,并能稳定表达HBsAg。
The recombinant plasmid pBIBSa containing the HBsAg DNA fragment was transferred into Agrobacterium tumefaciens strain LBA4404 directly. Ginseng cells were co-cultivated with Agrobacterium tumefaciens carrying pBIBSa. The ginseng cell lines carrying HBsAg-S gene were obtained. Transgenic cells were checked for the presence of target gene using PCR and RT-PCR. Samples containing the target gene showed a clear band at the site of 700 bp by agarose electrophoresis analysis (Figs.2, 3). Expressionlevels determined by ELISA showed maximum expression levels of 184 ng HBsAg/g FW and 0.009% of the total soluble protein. HBsAg in ginseng cells were located both on the membrane and in the nuclei (Fig.4).
出处
《植物生理与分子生物学学报》
CAS
CSCD
北大核心
2005年第5期551-554,共4页
Journal Of Plant Physiology and Molecular Biology
基金
吉林省科技发展计划项目(No.20030405)资助~~