摘要
为了获得口蹄疫病毒3AB基因重组伪狂犬病毒载体,试验以伪狂犬病毒TK/gⅠ缺失疫苗株为亲本株提取病毒基因组DNA,用限制性内切酶AscⅠ酶切基因组获得含完整PK基因的8.7 kb片段,将此片段克隆入pPolyⅡ载体的AscⅠ多克隆位点获得中间载体P8-AA;用限制性内切酶SacⅠ+NdeⅠ酶切质粒P8-AA,切去PK基因中的2 218 bp片段,在质粒P8-AA的PK基因缺失区插入已构建好的质粒pEGFP-3AB中含绿色荧光蛋白(EGFP)基因和3AB基因的完整表达盒,转染于猪肾细胞。结果表明:成功构建出可以用于同源重组的转移载体P8-EGFP-3AB,并且在猪肾细胞中成功表达了绿色荧光蛋白基因。
To construct recombinant Pseudorabies virus vector for 3AB gene of FMDV, the DNA from the viral genomes was extracted, taking Pseudorabies virus TK/g I gene - deleted strain as the parental strain, and the 8.7 kb fragment containing the whole PK geue was obtained using the digestion with Asc I . Plasmid P8 - AA was constructed by cloning the 8.7 kb fragment into the Asc I site of pPoly II. A total of 2 218 bp from the whole PK gene of plasmid P8 - AA was cut using the digestion with Sac I ＋ Nde I . The fragment from plasmid pEGFP - 3AB harboring the green fluorescent protein （EGFP） gene and the 3AB gene expression cassette was inserted into PK gene deletion region of the plasmid P8 -AA and then transfected into porcine kidney cells. The results showed that a transfer vector P8 -EGFP -3AB was constructed for homologous recombination, and GFP could be successfully expressed in porcine kidney cells.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2012年第6期15-18,182,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
辽宁省科技攻关项目(2007202006)
关键词
伪狂犬病毒
PK基因
口蹄疫病毒
3AB基因
Pscudorabies virus
protein kinase gene
Foot- and- mouth disease virus
3AB gene
