人呼吸道合胞病毒F1蛋白的原核表达及纯化

Prokaryotic Expression and Purification of F1 Protein of Human Respiratory Syncytial Virus

目的克隆A型人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)兰州分离株F1基因,并进行原核表达和纯化。方法采用RT-PCR方法克隆RSV F1 ORF序列,克隆至pET-42b(+)表达载体,构建重组原核表达质粒pET-42b-F1,转化大肠杆菌Rossata(DE3),IPTG诱导表达,表达产物经SDS-PAGE和Western blot鉴定后,用GST亲和层析树脂进行纯化。结果重组原核表达质粒经双酶切及测序证明构建正确;表达的融合蛋白相对分子质量约77 000,表达量约占菌体总蛋白的20%,以包涵体和可溶性两种形式存在;该蛋白可与鼠抗人RSV单抗发生特异性反应,纯化后纯度约为50%。结论已原核表达并纯化了HRSV F1蛋白,为其血清抗体的制备及免疫原性研究奠定了基础。

Objective To clone the F1 gene of Lanzhou isolate of human respiratory syncytial virus（HRSV） type A,express in prokaryotic cells and purify the expressed product.Methods The ORF sequence of HRSV F1 was amplified by RT-PCR and cloned into expression vector pET-42b（＋）.The constructed recombinant plasmid pET-42b-F1 was transformed to E.coli Rossata（DE3） for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by affinity chromatography with GST Binding Resin.Results Restriction analysis and sequencing proved that recombinant plasmid pET-42b-F1 was constructed correctly.The expressed fusion protein,with a relative molecular mass of about 77 000,contained about 20% of total somatic protein and existed in both soluble and inclusion body forms.The protein showed specific reaction with mouse anti-human RSV McAb,and reached a purity of about 50% after purification.Conclusion HRSV F1 protein was expressed in prokaryotic cells and purified,which laid a foundation of preparation and study on immunogenicity of serum antibody.