肠道病毒71型3C基因原核表达质粒的构建表达及纯化

Construction and expression of prokaryotic expression vector for 3C gene of enterovirus 71 and purification of expressed product

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 the prokaryotic expression vector for 3C gene of enterovirus 71（EV71）,express in E.coli and purify the expressed product.Methods The 3C gene of EV71 was amplified by PCR using the reverse transcription product of the virus as template,and cloned into vector pGEx-4T-1.The constructed recombinant plasmid pGEx-3C was transformed into E.coli BL21（DE3）for expression under induction of IPTG.The expressed recombinant fusion protein GST-3C was purified by GST-Agarose affinity chromatography and molecular sieve chromatography,and determined for reactogenicity by Western blot.Results Restriction analysis and sequencing proved the recombinant plasmid pGEx-3C was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of about 46 000,contained about 50% of total somatic protein,reached a purity of more than 90%,and showed specific binding to mouse monoclonal antibody against EV71.Conclusion Recombinant plasmid pGEx-3C was successfully constructed,and recombinant GST-3C fusion protein was highly expressed in secretory form in E.coli,which laid a foundation of study on structure and function of 3C protease of EV71.