摘要： 为了鉴定类猪圆环病毒P1 VP1蛋白能否在体外自组装成病毒样颗粒(VLPs),首先PCR扩增P1 ORF1 基因,利用pFastBac^TM1载体构建重组质粒,通过脂质体转染在Sf9细胞中表达P1 ORF1 基因。然后通过RT-PCR技术及Western Blot分别从转录和蛋白质水平上鉴定该基因的表达,应用透射电镜观察该蛋白质是否形成了VLPs。结果显示,转染Sf9细胞后,第5天开始出现细胞病变;从接毒细胞中提取总RNA,经Recombinant DNaseⅠ处理除去基因组DNA后,RT-PCR能够扩增出362 bp的目的片段;收获接毒后72 h的细胞,Western Blot分析显示,P1 ORF1 重组病毒样品能够与抗P1 VP1单抗发生特异性反应;电镜观察发现,该蛋白在Sf9细胞中形成了近椭圆形的包涵体。利用杆状病毒表达载体系统成功地在Sf9细胞中表达了类猪圆环病毒P1的 ORF1 基因,并证实在本试验条件下P1 VP1蛋白不能自组装成VLPs,为进一步研究P1 VP1蛋白的免疫学特性奠定基础。

关键词: P1 VP1, 昆虫杆状病毒表达载体系统, RT-PCR, Western Blot, 电镜

Abstract: To identify whether the porcine circovirus-like virus P1 VP1 protein can self-assemble into virus-like particles(VLPs) in vitro,P1 ORF1 gene was amplified and cloned into the pFastBac^TM1 vector.Then P1 ORF1 gene was expressed in Sf9 cells with liposome transfection method.The expression was identified on transcriptional and protein levels,respectively,using RT-PCR and Western Blot.The properties of P1 VP1 protein expressed here was observed using electron microscopy.The results showed that cytopathy on Sf9 cells appeared on the 5th day after transfection,the 362 bp target fragments was amplified by RT-PCR from total RNA extracted from the cell inoculation and treated by recombinant DNase I.And the results showed that the cell culture after 72 h of inoculation could work with monoclonal anti-P1 VP1 specific reaction with Western Blot,and the inclusion bodies oval-like formed by P1 VP1 protein in Sf9 cells was revealed with electron microscopy.Porcine circovirus-like P1 VP1 protein was successfully expressed with baculovirus expression vector system,and no VLPs were observed,which contributes to the further study of the immunological functions of P1 VP1 protein.

Key words: P1 VP1, Baculovirus expression vector system, RT-PCR, Western Blot, SEM

中图分类号: 

• S432.4