猪圆环病毒2型-伪狂犬重组病毒活疫苗SA215(C)株的构建

Construction and Characterization Analysis of the Recombinant Virus SA215(C) Strain of Pseudorabies Virus-Porcine Circovirus Type 2

根据GenBank发表的猪圆环病毒2型(PCV2)序列设计一对特异性引物,采用PCR方法,扩增PCV2ORF2基因,将ORF2基因插入到含有EGFP报告基因的转移载体质粒pPI-2.EGFP,获得重组中间转移质粒pPI-2.EGFP-ORF2。采用脂质体介导法,将重组中间转移载体pPI-2.EGFP-ORF2与伪狂犬病毒SA215株基因组共转染真核ST细胞,通过空斑纯化得到表达PCV2 ORF2基因和绿色荧光蛋白基因的重组伪狂犬病毒SA215(C)。经检测,重组病毒能表达具有生物活性的ORF2基因蛋白和绿色荧光蛋白。并且重组病毒及亲本株在同一细胞上的增殖滴度基本相同,表明EGFP和PCV2 ORF2基因的插入不影响PRV SA215的增殖。该重组病毒可作为猪圆环病毒2型和伪狂犬病毒的候选疫苗毒株。

ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 in GenBank. Following extraction and digestion, PCR products were subsequently inserted into universal transfer vector pPI-2. EGFP to generate re- combinant transfer plasmid pPI-2. EGFP-ORF2. The genomic DNA of PRV SA215 strain and pPI- 2. EGFP-ORF2 were co-transfected into ST cells with lipofectin,and recombinant virus SA215（C） was selected by fluorescence and PCR with ORF2 gene primers respectively. The recombinant virus was analyzed with Southern blotting and Western blotting. The results indicated that ORF2 gene of PCV2 had been inserted into the genome of PRV SA215 strain and the expressed EGFP- ORF2 fusion protein could react with PCV2 positive sera. Result of virus titers detection showed that the insertion of ORF2 gene did not influence propagation of recombinant virus.