猪瘟病毒Erns在杆状病毒系统中的表达和纯化

采用Bac-to-Bac表达系统构建重组杆状病毒rAcV-MBP-Erns,感染Sf9昆虫细胞,经免疫荧光及Western blot分析证实MBP-Erns融合蛋白在Sf9细胞中高效表达。表达的MBP-Erns以可溶和包涵体两种形式存在。在Sf9细胞中规模化增殖重组病毒,经Amylose Resin亲和层析纯化获得高纯度MBP-Erns,制备的MBP-Erns具有良好免疫原性,这些工作为研究该蛋白的生物学功能和免疫原性奠定基础。

Expression and Purification of Erns Glycoprotein of Classical Swine Fever Virus (CSFV)

To express the Erns protein of classical swine fever virus(CSFV),the Erns gene sequence of CSFV and the maltose binding protein(MBP) gene sequence were amplified by PCR.The PCR products were cloned into pFast HTa vector,respectively.The recombinant transfer plasmid pFast/MBP-Erns was identified by restriction enzyme and sequencing.After the transformation of DH10Bac-GFP cells by pFastMBP-Erns,recombinant bacmid MBP-Erns was produced.The MBP-Erns was transfected into Sf9 cells to generate the recombinant baculovirus,rAcV-MBP-Erns.Immunofluorescene and Western blot analysis confirmed that the recombinant MBP-Erns protein was successfully expressed in both soluble and inclusion body forms in Sf9 cells infected with rAcV-MBP-Erns.The high purity of the MBP-Erns protein was purified by affinity chromatography on amylose resin from large-scale Sf9 cell cultures.These studies contribute to our understanding of the biological function and immunogenicity of CSFV Erns.