目的真核表达人呼吸道合胞病毒(human respiratory syncytial virus,RSV)融合蛋白(fusion protein,F),并完成蛋白纯化及纯度测定。方法根据编码F蛋白的基因序列设计引物,PCR方法扩增出3′端带His标签的F基因序列,克隆入pGEM-T-easy载体,经核酸序列分析后,进一步克隆到pcDNA3.1(+)真核表达载体,限制性内切酶鉴定,用脂质体Lipofectamine 2000转染COS-7细胞,72h后再用Western blot检测目的蛋白的表达。Ni柱亲和层析纯化COS-7细胞表达的F蛋白,高效毛细管电泳分析纯化后蛋白纯度。结果核酸序列分析证实获得带His标签的RSVF基因序列,没有发生无义突变。转染COS-7细胞后,利用Western blot方法检测到F蛋白的特异性条带,纯度达99%以上。结论初步建立了真核表达RSVF蛋白的纯化方法,为进一步优化RSVF蛋白制备条件及单克隆抗体及诊断试剂等研究奠定了基础。 Objective To express fusion protein (F) of human respiratory syncytial virus (RSV) and to purify the protein and purify it. Methods According to the sequence of the gene encoding F protein, primers were designed. The 3 ’His-tagged F gene was amplified by PCR and cloned into pGEM-T-easy vector. After analysis by nucleic acid sequence, the F gene was further cloned into pcDNA3.1 (+ ) Eukaryotic expression vector and restriction endonuclease. Lipofectamine 2000 was used to transfect COS-7 cells. The expression of the target protein was detected by Western blot 72h later. Purification of F protein expressed by COS-7 cells by Ni column affinity chromatography and purified protein by high performance capillary electrophoresis. Results Nucleotide sequence analysis confirmed that the His-tagged RSVF gene sequence was obtained, and no nonsense mutation occurred. After transfection of COS-7 cells, the specific band of F protein was detected by Western blot, with a purity of more than 99%. Conclusion The purification method of eukaryotic expression of RSV F protein was preliminarily established, which laid the foundation for the further optimization of preparation conditions of RSV F protein, monoclonal antibody and diagnostic reagent.