猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)GP5蛋白是病毒主要囊膜结构蛋白和中和抗体主要靶蛋白,广泛应用于PRRS新型疫苗和诊断技术的研究.为获得具有与天然构象相近的PRRSV GP5蛋白,本研究根据草地贪夜蛾(Spdopterafrugiperda)密码子偏好性,优化PRRSV ORF5基因编码密码子,并克隆至杆状病毒载体后转染Sf9昆虫细胞,构建了重组杆状病毒rBac-PRRSV-GP5.经Western blot证实GP5蛋白在Sf9细胞中获得高效表达,且具有良好的免疫反应原性.经镍离子亲和层析纯化后,获得高纯度的重组GP5蛋白,为开展PRRSV诊断技术研发、生物学功能研究等奠定了抗原基础.
Expression and Characterization of Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein in Insect Baculovirus Expression System
Porcine reproductive and respiratory syndrome virus(PRRSV)GP5 protein is the main envelope structural protein of the virus and main target protein of neutralizing antibodies,which is widely used for the development of new vaccines and diagnostic approaches for PRRS.In order to maintain the natural structure of the PRRSV GP5 protein,the PRRSV ORF5 gene was optimized according to the codon preference of Spdopterafrugiperda,subsequently subcloned into the baculovirus vector and transfected into the Sf9 insect cells for expression of the recombinant rBac-PRRSV-ORF5.Western blot analysis showed that the GP5 protein was efficiently expressed in the Sf9 insect cells and exhibited high immunogenicity.The recombinant GP5 protein was further purified with nickel ion affinity chromatography.Our data laid a foundation for the biological study and development of diagnostic methods for PRRSV.
Porcine reproductive and respiratory syndrome virusORF5 geneGP5 proteinexpressionprotein purification
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