猪Ⅱ型圆环病毒-伪狂犬重组病毒活疫苗SA215(C)株的构建(初报)

根据GenBank发表的PCV2序列设计一对特异性引物,采用PCR方法,扩增PCV2 ORF2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,通过空斑纯化得到了表达猪Ⅱ型圆环病毒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Ⅱ型圆环病毒和伪狂犬病毒候选疫苗毒株。

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

ORF2 gene of porcine circovirus type Ⅱ were cloned by PCR with the specific pri mers de-signed according to genome of PCV2 in GenBank.Following extraction and digestion,PCR productswere subsequentlyinsertedinto universal transfer vector pPI-2.EGFPto generate recombinant trans-fer plasmid pPI-2.EGFP-ORF2.The genomic DNAof PRV SA215 strain and pPI-2.EGFP-ORF2 were co-transfectedinto STcells withlipofectin,and recombinant virus SA215(C) was selectedby fluorescence and PCR with ORF2 gene pri mers respectively.The recombinant virus was analyzedwith Southern blotting and Western blotting.The results indicates that the insertion of EGFP andPCV2 ORFS gene does not affect proliferation of PRVSA215.Such a group of virus can be chosen asthe alternative attenuated vaccine strainfor PCV-2 and PRV.Protein can react with PCV2 positivesera.Results of virustiters detectionshowtheinsertion of ORF2 gene does not influence propagation ofrecombinant virus.