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目的 开发简单、可靠的HTN、SEO型血清学分型检测方法。方法 应用RT -PCR法对全NP蛋白基因及其型特异区基因进行扩增 ,经TA克隆及Cui-SS、Cui- 15 5杆状病毒载体的构件、转座、获重组杆状病毒 ,再感染Sf9细胞进行表达。结果 克隆与表达了SEO病毒NP蛋白的全蛋白编码区及型特异编码区 (15 5到 4 2 9氨基酸处的编码基因 ) ,全NP蛋白表达产物与病毒株感染VeroE6细胞的反应谱完全一致 ,型特异区表达产物与相应的型特异McAb结合。结论 建立了SEO型病毒全NP蛋白及NP蛋白型特异区编码基因的克隆和表达方法 ,为开发简便、可靠的SEO型血清学分型检验方法奠定了基础。
Objective To develop a simple and reliable HTN and SEO serological typing assay. Methods The whole NP protein gene and its type-specific region gene were amplified by RT-PCR. After TA cloning and construction of Cui-SS and Cui-15 baculovirus vectors, recombinant baculovirus was obtained. Sf9 cells infected for expression. Results The whole protein coding region and the type-specific coding region (15 to 429 amino acid coding genes) of the SEO virus NP protein were cloned and expressed. The full NP protein expression product was completely consistent with that of the virus strain infected VeroE6 cells. Type specific region of the expression product with the corresponding type-specific McAb. Conclusion The methods of cloning and expressing full-NP of SEO virus and coding region of NP protein-specific region were established, which laid the foundation for the development of a simple and reliable SEO serotyping test.