表达猪Ⅱ型圆环病毒ORF2基因重组鸡痘病毒的构建与鉴定

目的构建表达猪Ⅱ型圆环病毒(PCV2)ORF2基因的重组鸡痘病毒。方法将PCV2ORF2基因插入到转移载体pUTAL复合启动子下游,构建重组鸡痘病毒转移质粒pUTA-ORF2,将该质粒与鸡痘病毒野生株282-E4共转染鸡胚成纤维细胞(CEF),进行同源重组。经BrdU加压筛选重组鸡痘病毒,采用RT-PCR和间接免疫荧光法进行鉴定。结果重组鸡痘病毒感染的CEF经RT-PCR可扩增出260bp的目的基因,间接免疫荧光检测可见特异性黄绿色荧光,表明目的基因在重组鸡痘病毒中得到表达。结论已成功构建了1株表达PCV2ORF2基因的重组鸡痘病毒。

Construction and Identification of Recombinant Fowlpox Virus Expressing ORF2 Gene of Porcine Circovirus Type 2

Objective To construct the recombinant fowlpox virus expressing the ORF2 gene of porcine circovirus type 2 (PCV2). Methods The ORF2 gene of PCV2 was inserted downstream to the complex promoter of transfer vector pUTAL, and the constructed recombinant plasmid pUTA-ORF2 was co-transfected to chick embryo fibroblasts (CEFs)with wild fowlpox virus strain 282-E4 for homologous recombination. The recombinant fowlpox virus was screened by BrdU pressure screening and identified by RTPCR and indirect IFA. Results The target gene at a length of 260 bp was amplified from CEFs transfected with recombinant fowlpox virus. Specific yellowish-green fluorescence was observed in the CEFs transfected with recombinant fowlpox virus, indicating expression of target gene. Conclusion A recombinant fowlpox virus strain expressing the ORF2 gene of PCV2 was successfully constructed.