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目的构建肠道病毒71型(Entervirus71,EV71)3C基因的原核表达质粒,在大肠杆菌中进行表达并纯化。方法以病毒的反转录产物为模板,PCR扩增EV71 3C基因,克隆至pGEx-4T-1载体中,构建重组表达质粒pGEx-3C,转化大肠杆菌BL21(DE3)中,IPTG诱导表达。表达的重组融合蛋白GSR-3C经GST-Agarose亲和层析和分子筛层析纯化后,Western blot检测其反应原性。结果重组表达质粒pGEx-3C经双酶切及测序证实构建正确;表达的重组融合蛋白相对分子质量约46 000,表达量约占菌体总蛋白的50%,主要以可溶性形式表达;纯化后的重组融合蛋白纯度大于90%,可与小鼠抗EV71单克隆抗体特异性结合。结论成功构建了重组表达质粒pGEx-3C,在大肠杆菌中高效分泌表达并纯化了重组GST-3C融合蛋白,为EV713C蛋白酶结构与功能的研究及3C靶向药物和疫苗的研究奠定了基础。
Objective To construct a prokaryotic expression plasmid of 3C gene of Enterovirus 71 (EV71), which was expressed in Escherichia coli and purified. Methods The EV71 3C gene was amplified by PCR using the reverse transcription product of the virus as a template and cloned into pGEx-4T-1 vector. The recombinant plasmid pGEx-3C was constructed and transformed into E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombinant fusion protein GSR-3C was purified by GST-Agarose affinity chromatography and molecular sieve chromatography, and its reactivity was determined by Western blot. Results The recombinant plasmid pGEx-3C was confirmed by double enzyme digestion and sequencing. The recombinant fusion protein was about 46 000 in molecular weight and accounted for about 50% of the total bacterial proteins, which was mainly expressed in soluble form. After purification Recombinant fusion protein purity greater than 90%, with mouse anti-EV71 monoclonal antibody specific binding. Conclusion The recombinant plasmid pGEx-3C was successfully constructed and secreted and purified in Escherichia coli. Recombinant GST-3C fusion protein was successfully constructed, which laid the foundation for the study of the structure and function of EV713C protease and the research of 3C targeted drugs and vaccines.