乙肝病毒表面抗原基因在人参细胞中的表达

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HBsAg184 ng,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.

Expression of Hepatitis B Surface Antigen Gene in Ginseng Cell

The recombinant plasmid pBIBSa contain- ing 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 us- ing PCR and RT-PCR. Samples containing the target gene showed a clear band at the site of 700 bp by aga- rose electrophoresis analysis (Figs.2, 3). Expressionlevels determined by ELISA showed maximum expres- sion levels of 184 ng HBsAg/g FW and 0.009% of the total soluble protein. HBsAg in ginseng cells were lo- cated both on the membrane and in the nuclei (Fig.4).