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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 ,为进一步研究食用疫苗开辟了新途径
Advances in the field of genetic engineering make plants a medicinal protein production system with important economic value. The full-length 2.2 kb capsid protein fusion gene sequence was obtained by PCR amplification with a specific primer for the hepatitis A virus capsid protein fusion gene using the cloning vector pCDNAⅡA16 containing the structural gene cDNA of hepatitis A virus as a template. After sequencing, it was cloned into the plant expression vector pBI12 1, and the coat protein fusion gene was located in the left and right border of pBI12 1 plasmid T DNA under the control of CaMV35S promoter. After restriction endonuclease analysis and PCR identification, the recombinant plasmid pBI12 1 A was introduced into Agrobacterium tumefaciens LBA4404 by freeze-thaw method. Using the hypocotyls of Citrus sinensis Osbeck as the transformation material, the capsid protein fusion gene was transformed into plant genome by Agrobacterium-mediated transformation. 12 0 transformed explants were screened by kanamycin 50mg / L, of which 13 were in good condition and no albino phenomenon. PCR analysis showed that 5 of the 13 plants to be transformed had been introduced into the hepatitis A virus capsid protein fusion gene with a conversion rate of 41%. This study is a preliminary study on the expression of foreign proteins in genetically transformed citrus, which opens up new ways for further research on the edible vaccine