| 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-mouth disease virus,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3.57 g/L,而根中可溶性蛋白含量最低,仅有0.17 g/L;靠近心叶的叶片可溶性蛋白含量可达到1.475 g/L,而最外部叶片可溶性蛋白含量仅有0.37 g/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。 |
| 关键词: 转基因胡萝卜 口服疫苗 口蹄疫病毒VP1基因 |
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| Expression of foot-and-mouth disease virus VP1 gene in carrots |
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| Abstract: |
| 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. |
| Key words: transgenic carrots,oral vaccine,FMDV VP1 gene |
