猪波形蛋白对PRRSV感染Marc-145细胞的抑制作用

为了研究波形蛋白在介导猪繁殖与呼吸综合征病毒(PRRSV)感染细胞过程中的作用,利用RT-PCR方法从PRRSV非易感细胞系猪肾细胞系PK-15细胞中扩增目的基因,克隆入pET-28a(+)载体,转化大肠杆菌BL21(DE3)中进行诱导表达。表达的重组猪波形蛋白经SDS-PAGE和Western blot鉴定和纯化后免疫新西兰大白兔制备多克隆抗体。利用病毒抑制试验检测重组猪波形蛋白及多克隆抗体在PRRSV感染Marc-145细胞过程中的作用。结果表明,成功扩增猪波形蛋白基因并克隆入pET-28a载体,经诱导后得到高效表达,纯化后免疫兔子产生高价血清抗体(105)。病毒阻断结果表明,猪波形蛋白及多克隆抗体均能阻断PRRSV感染Marc-145细胞。这为以波形蛋白为基础的PRRSV受体阻断抑制剂的研究提供了实验依据。

Role of Swine Vimentin in PRRSV Infection to Marc-145 Cells

To investigate the function of swine vimentin in PRRSV infection,the swine vimentin gene from a PRRSV non-susceptible cell lines PK-15 cells was amplified by RT-PCR and cloned into pET-28a vector.The recombinant swine vimentin protein was expressed in Escherichia coli BL21(DE3) and was identified by SDS-PAGE and Western blot.After purification,the target protein was used to immunize New Zealand rabbit to produce polyclonal antibodies.The recombinant swine vimentin protein and polyclonal antibodies were used to test blocking function of PRRSV infection to Marc-145 cells by virus inhibition.The results showed that the swine vimentin gene was amplified successfully from PK-15 cells and cloned into pET-28a vector.The rabbit anti-vimentin antibodies were produced with the titer of 1×105.PRRSV infection to Marc-145 cells was blocked partially by the recombinant vimentin protein while blocked completely by the antibodies.These results provide additional information for the research of PRRSV inhibitor based on vimentin.