论文部分内容阅读
构建HBV表面抗原 (HBsAg)植物表达载体并在胡萝卜植株中表达。采用自行构建adr亚型HBsAg基因克隆T adr,再次酶切获得 860bp含PreS2的HBsAg基因片段 ,将其插入到植物表达载体pBPC5 5 ,新质粒命名为pBPC91adr。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒 pBPC93共同经基因枪 (PDS 10 0 0 /He)转化胡萝卜悬浮细胞 ,经含除草剂 (Biolaphos)的培养基筛选及植物激素诱导分化 ,获得除草剂抗性胡萝卜幼苗。结果为转化后 8周 ,自胡萝卜细胞中分化出除草剂抗性胡萝卜幼苗。提取新分化幼苗总DNA ,特异性引物PCR扩增后可见860bp扩增带 ;Southern Blot证明有HBsAg基因整合 ,胡萝卜蛋白萃取物的Western Blot及ELISA检测证实有HBsAg蛋白表达。利用基因枪转化使质粒 pBPC91adr中HBsAg基因在胡萝卜幼苗内整合并表达 ,提示以植物生产疫苗具有可行性
A HBV surface antigen (HBsAg) plant expression vector was constructed and expressed in carrot plants. The HBsAg gene fragment containing 860bp PreS2 was digested by restriction endonuclease T adr. The inserted fragment was inserted into the plant expression vector pBPC5 5, and the new plasmid was named pBPC91adr. The transgenic carrot cells were transformed with the plasmid pBPC93 containing the herbicide resistance gene and the GUS protein gene into the carrot suspension cells by means of a gene gun (PDS10 0 / He), followed by screening with a medium containing the herbicide (Biolaphos) and plant hormone-induced differentiation To obtain herbicide-resistant carrot seedlings. As a result, 8 weeks after the transformation, the herbicide-resistant carrot seedlings were differentiated from the carrot cells. The total DNA of the newly differentiated seedlings was extracted. The 860bp amplified bands were obtained after PCR amplification of the specific primers. The integration of HBsAg gene was confirmed by Southern Blot, and the expression of HBsAg protein was confirmed by Western Blot and ELISA of carrot protein extracts. The use of gene gun transformation in the plasmid pBPC91adr HBsAg gene integration and expression in carrot seedlings suggest that the feasibility of plant-based vaccines