波形蛋白介导猪繁殖与呼吸综合征病毒感染Marc-145细胞作用的初步研究  

The Role of Vimentin during PRRSV Infection of Marc-145 Cells

作　　者：王伟伟[1] 张璐[1] 马晓春[1] 高继明[1] 肖一红[1] 周恩民[2] 

机构地区：[1]山东农业大学动物医学院,山东泰安271018 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《病毒学报》2011年第5期456-461,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(30871857;30901063;U0931003/L01);高等学校博士学科点专项科研基金(20093702120004)

摘　　要：为了研究波形蛋白在介导PRRSV感染过程中的作用,根据GenBank中已发表的波形蛋白序列,设计特异性引物,利用RT-PCR方法从Marc-145细胞中扩增目的基因,克隆入pET-28a,转化表达菌BL21(DE3)中进行诱导表达,用SDS-PAGE和Western blot鉴定表达产物,将表达产物纯化后免疫BALB/c小鼠制备血清,用病毒阻断实验验证PRRSV与波形蛋白及其抗体的关系。用ELISA方法确定了重组波形蛋白与PRRSV结构蛋白N蛋白和囊膜蛋白GP5蛋白之间的关系。结果表明,成功的从Marc-145细胞中扩增出全长的波形蛋白基因,将其克隆入pET-28a,诱导后得到高效表达,表达蛋白纯化后免疫小鼠抗体效价达到105,病毒阻断实验表明波形蛋白能部分阻断PRRSV感染,其抗血清能完全阻断PRRSV感染。ELISA结果表明波形蛋白能与N蛋白结合而不与GP5蛋白结合。此结果为PRRSV感染的细胞的受体机制增添了新的内容,为PRRSV感染过程中受体间的相互作用关系研究奠定基础。The objective of this study was to investigate the function of vimentin in PRRSV infection. Vimentin gene from Marc-145 cells was amplified by RT-PCR, cloned into pET-28a vector and expressed in Escherichia coli BL21（DE3）. The expressed vimentin was confirmed by Western blot and purified which was used to immunize BALB/c mice for the production of antibodies. Vimentin and antibodies were tested for blocking PRRSV infection of Marc-145 cells. The binding of vimentin to PRRSV N and GP5 proteins were tested by the ELISA. The results showed that vimentin gene was amplified successfully and expressed as identified by SDS-PAGE and Western blot. Mouse anti-vimentin antibodies were produced with the titer of 105. PRRSV infection of Marc-145 cells was blocked partially by vimentin while blocked completely by the antibobies. In addition, vimentin was bound N protein, but not GPS. These results provide additional information on PRRSV entry into Marc-145 cells.

关 键 词：猪繁殖与呼吸综合征病毒 波形蛋白 表达 病毒阻断 N蛋白 

分 类 号：S852.4[农业科学—基础兽医学]