【摘要】 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。更多还原

【Abstract】 Objective To express and purify the fusion protein(F)of human respiratory syncytial virus(RSV)for application in diagnosis and vaccine.Methods F gene with 3’ end thrombin cleavage site and six histidine tags was obtained by PCR.The resultant F-His gene was subcloned into pGEM-T-easy vector.After nucleotide sequence analysis,F-His gene was cloned into eukaryotic expression vector pcDNA3.1(+),and then transfected into COS-7 cells by using Lipofectamin 2000.F-His protein in the lysatewas was detected by Western blot assay at 72 hours posttransfection,and then purified by Ni2+-affinity chromatograph.The purified F-His protein was analyzed by high performance capillary electrophoresis(HPCE).Results DNA sequencing displayed no nonsense mutation in amplified F-His gene.The specific expression of F-His protein in COS-7 cells was confirmed by Western blot assay and the purity of purified F-His protein was more than 99%.Conclusion The established method can be used to prepare RSV F protein for further investigation on monoclonal antibody and diagnostic kit.更多还原