尼帕病毒融合蛋白的真核表达及其特异性血清的制备

Eukaryotic Expression of Nipah Virus Fusion Protein and Preparation of its Specific Antiserum

目的:用真核细胞表达可溶性尼帕病毒融合蛋白(sF),通过免疫小鼠获得抗F蛋白的特异性血清。方法:设计构建重组质粒pcDNA-sF,在哺乳动物表达系统中进行表达,通过Strep柱亲和层析纯化目的蛋白,Western印迹检测目的蛋白,免疫小鼠后制备抗F蛋白的特异性血清,并通过ELISA鉴定抗血清的结合活性。结果:尼帕病毒F蛋白在哺乳动物细胞中获得可溶性表达,纯化得到的目的蛋白大小正确,胰蛋白酶切鉴定其具有与天然蛋白相似的切割产物;制备的抗血清能特异性结合目的蛋白。结论:获得了可溶性尼帕病毒融合蛋白及高效价抗F蛋白的特异性血清,可用于尼帕病毒疫苗评价、特异性抗体筛选等相关研究。

Objective:To express soluble form of Nipah virus fusion protein(sF)in eukaryotic cells,and to produce its specific serum against the F protein by immunizing mice.Methods:The recombinant plasmid pcDNA-sF was constructed and transfected into Expi293F cells.The protein of interest was purified through Strep tag affinity chromatography.The purified protein was identified by Western blotting.The anti-F specific serum was prepared by immunizing mice and detected antiserum binding activity by ELISA.Results:We obtained the sF protein from mammalian cells,and the purified protein was of the correct size and was cleaved by trypsin similar to native fusion protein;the prepared antiserum can specifically bind to the target protein.Conclusion:The sF and high titer anti-F protein was obtained,and it can be used for Nipah virus vaccine evaluation,specific antibody screening and other related research.