Wang Wei-Wei, Zhang Lu, Ma Xiao-Chun, Gao Ji-Ming, Xiao Yi-Hong, Zhou En-Min
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, TaiAn, Shandong 271018, China.
Bing Du Xue Bao. 2011 Sep;27(5):456-61.
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 10(5). 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 GP5. These results provide additional information on PRRSV entry into Marc-145 cells.
本研究的目的是探讨波形蛋白在猪繁殖与呼吸综合征病毒（PRRSV）感染中的作用。通过逆转录聚合酶链反应（RT-PCR）从Marc-145细胞中扩增波形蛋白基因，将其克隆到pET-28a载体中并在大肠杆菌BL21(DE3)中表达。通过蛋白质免疫印迹法（Western blot）确认表达的波形蛋白并进行纯化，用于免疫BALB/c小鼠以产生抗体。检测波形蛋白和抗体对Marc-145细胞PRRSV感染的阻断作用。通过酶联免疫吸附测定（ELISA）检测波形蛋白与PRRSV N蛋白和GP5蛋白的结合。结果显示，波形蛋白基因成功扩增，并经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法鉴定表达成功。产生了效价为10(5)的小鼠抗波形蛋白抗体。波形蛋白部分阻断Marc-145细胞的PRRSV感染，而抗体则完全阻断。此外，波形蛋白与N蛋白结合，但不与GP5结合。这些结果为PRRSV进入Marc-145细胞提供了更多信息。
