真核表达人呼吸道合胞病毒F蛋白的纯化方法

Purification of the fusion protein of human respiratory syncytial virus expressed in eukaryotic expression vector

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 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 subcloncd 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 Ni^2＋ - affinity chromatograph. The purified F-His protein was analyzed by high performance capilhuT 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 RST F protein for further investigation on monoclonal antibody and diagnostic kit.