摘要
分别用限制性核酸内切酶EcoRⅠ、BamHⅠ和KpnⅠ酶切含PRVFa株gI片段的质粒pPI,补平连接后得到去除EcoRⅠ、HindⅢ、EcoRⅤ、BamHⅠ和KpnⅠ酶切位点的大小约5.7kb的质粒pPI-2,再以CMV早期启动子控制的增强型绿色荧光蛋白(EGFP)基因作为报告基因,将其插入pPI-2中gI基因起始密码子ATG下游的StuⅠ位点,构建了以gI为同源序列含有EGFP报告基因的转移载体质粒pPI-2.EGFP。然后根据AnaI.Ranz等报道的猪细小病毒(PPV)NADL-2株的序列,设计一对包含其结构蛋白VP2基因的PCR引物,扩增得到VP2基因后,将其插入转移载体质粒pPI-2.EGFP中,首次构建了含PPVVP2基因及其EGFP报告基因的PRV基因缺失转移载体质粒pPI-2.EGFP.VP2,为进一步构建PRV重组PPVVP2基因活载体疫苗奠定了基础,同时EGFP的引入也为进一步研究PRV在机体内的定植和感染机理打下了基础。
By means of restriction enzyme digestion, blunting and ligation the plasmid pPI-2 of Pseudorabies virus Fa strain was obtained based on plasmid pPI deleted restriction enzyme sites EcoRⅠ, HindⅢ, EcoRⅤ, BamHⅠ and KpnⅠ. After digestion with ApaLⅠ and MluⅠ, the reporting gene EGFP from plasmid pEGFP-C1 was inserted into the StuⅠ site downstream of initiation codon ATG of gI gene, the transfer vector pPI-2.EGFP was constructed. A pair of primers of VP2 gene was designed according to PPV genomic sequence reported by Ana I.Ranz. The VP2 gene obtained by PCR was inserted into pPI-2.EGFP vector, and the transfer vector pPI-2.EGFP.VP2 was then constructed. By using the methods of Dot-blots hybridization and restriction enzymes digestion, the results suggest that the construction of pPI-2.EGFP.VP2 is successful. The successful construction of pPI-2.EGFP.VP2 is to lay a foundation on the development of an genetically engineered PRV bivalent marker vaccine.
出处
《四川农业大学学报》
CSCD
2005年第2期232-237,共6页
Journal of Sichuan Agricultural University
基金
国家"863"项目
教育部科研项目
四川省科技厅重点攻关项目。
