摘要
为了生产一种可以直接口服的口蹄疫疫苗，以胡萝卜（Daucus carota L．var．sativa）无菌幼苗下胚轴诱导的愈伤组织为受体材料，通过农杆菌LBA4404介导的遗传转化，在CaMV双35S启动子的驱动下，将口蹄疫病毒（foot-and-mouthd iseasevirus，FMDV）结构蛋白VP1基因的活性肽片段（包括2个141～160位肽和1个200-213位肽的基因片段）导入胡萝卜愈伤组织细胞。经PCR和PCR-Southern杂交等方法鉴定转化苗，确认口蹄疫病毒结构蛋白VP1基因已整合到胡萝卜染色体中，转化效率达到52％。SDS-PAGE试验结果初步证明，在转化苗总蛋白中有目的基因表达，胡萝卜种子中可溶性蛋白含量最高，达3．57g／L，而根中可溶性蛋白含量最低，仅有0．17g／L；靠近心叶的叶片可溶性蛋白含量可达到1．475g／L，而最外部叶片可溶性蛋白含量仅有0．37g／L，相差近5倍。Dot-ELISA和ELISA检测结果表明，转化苗中表达的口蹄疫病毒结构蛋白VP1可以与FMDV灭活疫苗免疫动物制备的抗体特异结合。以上试验结果证明，口蹄疫病毒结构蛋白VP1基因在转基因胡萝卜中表达成功。
In order to produce a kind of oral FMDV vaccine,plant expression vector pBFMDV containing VP1 gene fragments （containing two 141-160 peptides and one 200-213 peptide） of foot-and-mouth disease virus （FMDV） under the control of cauliflower mosaic virus （CaMV） 35S double promoters was constructed. By infected with Agrobacterium tumefaciens LBA4404,the gene fragments were transferred into carrot calli. PCR and PCR-Southern analyses of transgenic carrot DNA confirmed that the fragments of FMDV VP1 gene were introduced into the plant genomic DNA successfully,the rate of transformation was up to 52%. The result of SDS-PAGE showed that the gene fragments of interest were expressed in some of the transgenic plants. The soluble protein from carrot＇s seed is the highest,and the quantity is up to 3.57 g/L. However,the protein from carrot＇s root is the lowest ,and the quantity is only 0. 17 g/L. What＇s more, the soluble protein from apical leaf is up to 1. 475 g/L,but the protein from outward leaf is only 0.37 g/L. The difference between the two was more than 5-fold. The binding activity of the expressed proteins to anti-FMDV antibodies was confirmed by using Dot-ELISA and ELISA. These results demonstrated that the fragments of FMDV VP1 gene had been expressed in transgenic carrots successfully.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2007年第3期75-80,共6页
Journal of Northwest A&F University(Natural Science Edition)
