密码子优化型鹅细小病毒病毒样颗粒的表达与鉴定

根据昆虫细胞密码子的偏嗜性,对鹅细小病毒(Goose parvovirus,GPV)VP2基因的密码子进行优化,利用Bac-to-Bac表达系统构建了表达优化VP2基因的重组杆状病毒,通过感染昆虫细胞(Sf9)获得病毒样颗粒(virus-like particles,VLPs)。SDS-PAGE分析显示Sf9细胞中出现了65 kDa的蛋白条带,间接免疫荧光(indirect immunofluorescent assay,IFA)和Western blot结果均证实表达产物能与鼠抗GPV VP2抗体发生特异性反应,说明其具有良好的免疫反应性。同时,试验结果还证明,密码子优化后的GPV VP2基因在昆虫细胞中表达量得到了提高,明显高于野生型VP2基因。透射电镜观察纯化后的昆虫细胞表达产物,可见直径约30 nm的VLPs,表明密码子的优化并不影响VP2蛋白的组装。本研究为进一步研制诊断抗原以及新型GPV疫苗等奠定了基础。

CHARACTERIZATION OF VIRUS-LIKE PARTICLES DERIVED FROM CODON-OPTIMIZED GOOSE PARVOVIRUS VP2 GENE PRODUCTS EXPRESSED IN INSECT CELLS

To enhance the expression level of Goose parvovirus(GPV) VP2 through baculovirus expression system(BES),the codon usage of GPV VP2 gene was optimized according to the codon bias of insect cells.The recombinant GPV with codon-optimized VP2 gene was then used to infect Sf9 insect cells.The expressed products were characterized in SDS-PAGE,Western blot and indirect immunofluorescence assay(IFA).A protein of molecular mass of 65 kDa reacted specifically with GPV VP2 antiserum.Immunofluorescent staining showed that codon-optimized VP2 was expressed in Sf9 cells.The virus-like particles(VLPs) of about 30 nm in diameter were observed under electron microscopy.The expression level of codon-optimized VP2 was significantly higher than the wild type.These findings indicated that VLPs derived from codon-optimized GPV VP2 products expressed in Sf9 insect cells yielded were useful for development of diagnostic reagents and vaccines.