摘要
基因工程领域的研究进展使得植物体成为具有重要经济价值的药用蛋白的生产体系。以含甲型肝炎病毒结构基因cDNA的克隆载体 pCDNAⅡA16为模板 ,用甲型肝炎病毒衣壳蛋白融合基因特异引物进行PCR扩增 ,得到全长 2 .2kb衣壳蛋白融合基因序列。经测序鉴定后正向克隆于植物表达载体 pBI12 1中 ,衣壳蛋白融合基因位于pBI12 1质粒T DNA左右边界区间内 ,处于CaMV35S启动子控制之下。经限制性内切酶分析和PCR鉴定后利用冻融法将重组质粒 pBI12 1 A导入根癌农杆菌LBA4 4 0 4。以锦橙 (Citrus.SinensisOsbeck)上胚轴为转化材料 ,通过根癌农杆菌介导法将衣壳蛋白融合基因转化到植物基因组中。 12 0株转化外植体经卡那霉素 5 0mg/L筛选 ,其中 13株生长状况良好未出现白化现象的拟转化芽微嫁接到实生砧木继续培养。PCR分析证明 ,13株拟转化植株中有 5株植物基因组中已导入甲型肝炎病毒衣壳蛋白融合基因 ,转化率为 4 1%。此研究是对遗传转化柑桔表达外源蛋白的初步探讨 。
The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments. 13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.
出处
《遗传》
CAS
CSCD
北大核心
2004年第4期425-431,共7页
Hereditas（Beijing)
基金
重庆市科委应用基础研究项目资助~~
