猪圆环病毒2型DY株ORF2基因在Sf9昆虫细胞中的表达

本试验采用PCR方法扩增出完整的PCV2 ORF2基因,并将其克隆到Bac-to-Bac杆状病毒表达系统的转移载体pFastBac1^TM中,获得重组转移载体pFast-ORF2,再将其转化DH10Bac感受态细胞,发生转座反应,通过抗性及蓝白斑筛选获得含有ORF2基因的重组杆粒,通过Cellfectin转染试剂介导将重组杆粒DNA转染Sf9昆虫细胞,获得重组杆状病毒。用该重组杆状病毒感染Sf9昆虫细胞,并通过间接免疫荧光法和Western blotting检测目的蛋白的表达。结果表明,ORF2基因在昆虫细胞中获得表达,表达的蛋白质可被PCV2阳性血清识别,这为进一步研究PCV2亚单位疫苗及诊断抗原奠定了基础。

Expression of ORF2 Gene of Porcine Circovirus Type 2 in Sf9 Insect Cells

The ORF2 gene of porcine circovirus type 2(PCV2) including complete open reading frame(ORF)was amplified by PCR,and it was cloned into the pFastBac1^TM vector of Bac-to-Bac baculovirus expression systems to construct the recombinant transfer vector pFast-ORF2.The transfer vector was transformed into DH10Bac competent cell which contained the BmNPV bacmid. A recombinant shuttle vector which contained ORF2 gene was constructed by site-specific transposition and the colonies containing recombinant bacmid were collected by white selection. The cultured Sf9 cells were transfected with recombinant bacmid DNA mediating with cellfectin reagent and the pure recombinant baculovirus Bacmid-ORF2 was obtained. After transfecting this transformant into Sf9 cells,Cap protein expression in Sf9 cells was detected by indirect immunofluorescent assay and Western blotting. The experimental results showed that the recombinant baculovirus was obtained and the Cap protein with a molecular weight of 31 ku was expressed in insect Sf9 cells. The recombinant protein could be recognized by PCV2 positive serum. This would lay a foundation for developing PCV2 subunit vaccine and diagnosis method.