表达PRRSV ORF5和PCV2 ORF2基因的重组鸡痘病毒的构建及鉴定

Construction of recombinant fowl pox virus expressing ORF5 of PRRSV and ORF2 of PCV-2

分别将猪圆环病毒2型的ORF2基因和猪繁殖与呼吸综合征病毒的ORF5基因,插入到鸡痘病毒转移载体pUTA2-16-LacZ单一启动子和复合启动子下游,构建重组鸡痘病毒转移质粒pUTAL-ORF5-ORF2。将该重组质粒与鸡痘病毒282-E4株共转染鸡胚成纤维细胞,进行同源重组。通过3次溴脱氧尿嘧啶核苷(BrdU)加压筛选,经RT-PCR、IFA和Western blot鉴定,表明重组鸡痘病毒中的目的基因在鸡胚成纤维细胞中得到表达,获得一株携带有目的基因ORF2和ORF5的重组鸡痘病毒rFPV-ORF2-ORF5。

Recombinant plasmid pUTAL-ORF2-ORF5 was constructed by cloning the ORF2 gene of PCV2 and the ORF5 gene of PRRSV into the fowl pox virus transfer vector pUTA2-16LacZ under single promoter and composite promoter, respectively. Chick embryo fibroblast cell （CEF） was co-transfected with plasmid pUTAL-ORF2-ORF5 and FPV 282-E4 strain for homologous recombination. Recombinant virus was screened 3 times in the presence of 5＇-bromo-deoxyuridine （BrdU） and identified by RT-PCR, IFA and Western blot. Results showed that the target gene in recombinant fowl pox virus was efficiently expressed in CEF. A recombination fowl pox virus strain rFPV-ORF2-ORF5 expressing ORF2 gene of PRRSV and the ORF5 gene of PCV2 was successfully constructed.