Sunil Kumar G B, Ganapathi T R, Revathi C J, Prasad K S N, Bapat V A
Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Protein Expr Purif. 2003 Nov;32(1):10-7. doi: 10.1016/j.pep.2003.07.004.
Hepatitis B virus ' s ' gene coding for surface antigen was cloned into plant transformation vectors pHER100 and pHBs100 with and without endoplasmic reticulum retention signal, respectively. Transformed tobacco cell lines were analyzed for the integration of the transgene by PCR and Southern blot hybridization. Expression levels as determined by ELISA showed maximum expression levels of 2 microg HBsAg gm(-1) fresh weight and 10 ng mL(-1) of spent medium in pHER100 transformed cells. Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed cells. HBsAg was expressed both as intracellular and secreted forms in pHER100 transformed cells. The buoyant density in CsCl of HBsAg derived from pHBs100 transformed tobacco cells was determined and found to be 1.095 g mL(-1). HBsAg obtained from transformed tobacco cells is similar to the human serum derived one in buoyant density properties. This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium.
将编码表面抗原的乙肝病毒“s”基因分别克隆到带有和不带有内质网滞留信号的植物转化载体pHER100和pHBs100中。通过PCR和Southern印迹杂交分析转化的烟草细胞系中转基因的整合情况。ELISA测定的表达水平显示，在pHER100转化的细胞中，最大表达水平为每克鲜重2微克乙肝表面抗原（HBsAg）和每毫升消耗培养基10纳克。蛋白质印迹分析证实，在转化细胞中存在特异于HBsAg的24 kDa条带。在pHER100转化的细胞中，HBsAg以细胞内和分泌形式表达。测定了来自pHBs100转化烟草细胞的HBsAg在氯化铯中的浮力密度，发现为1.095克/毫升。从转化烟草细胞获得的HBsAg在浮力密度特性上与人血清来源的HBsAg相似。这是关于植物细胞将HBsAg颗粒分泌到细胞培养基中的首次报道。
