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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 F1 gene of Lanzhou strain of human respiratory syncytial virus (HRSV), and to express and purify it. Methods The ORF sequence of RSV F1 was cloned by RT-PCR and cloned into pET-42b (+) expression vector. The recombinant prokaryotic expression vector pET-42b-F1 was constructed and transformed into E.coli Rossata (DE3) After identification by PAGE and Western blot, purification was performed with GST affinity chromatography resin. Results The recombinant prokaryotic expression plasmid was proved by double enzyme digestion and sequencing. The relative molecular mass of the expressed fusion protein was about 77 000, and the expression level was about 20% of the total bacterial protein. The fusion protein existed in both inclusion body and soluble form. It can react specifically with mouse anti-human RSV monoclonal antibody and the purity is about 50% after purification. Conclusion The prokaryotic expression and purification of HRSV F1 protein has laid the foundation for the preparation of its serum antibody and its immunogenicity.