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目的 构建泡球蚴18(Em18)基因的原核表达质粒,获得高效表达、有生物活性的Em18重组蛋白,为包虫病诊断试剂的研制奠定基础。 方法 用DNAman软件设计引物,分别在引物5′端和3′端添加EcoRⅠ和 XhoⅠ酶切位点,以pMD18 T/Em18原核表达质粒为模板,PCR扩增Em18基因片断,经酶切,克隆入原核表达载体 pET 41a(+),构建原核表达质粒 pET41a Em18;转化大肠埃希菌BL21(DE3),酶切、PCR及测序鉴定其插入序列的正确性。经 IPTG诱导表达 rEm18 GST重组蛋白和GST重组蛋白,用谷光甘肽 Sepharose 4B亲合层析柱分别进行纯化,通过 SDS PAGE和Western blot试验分析鉴定。 结果 测序表明构建的 pET41a Em18 原核表达质粒均为正确连接,插入 Em18 基因片断为486 bp。SDS PAGE检测表明Em18基因以 rEm18 GST重组蛋白的方式得到成功表达,在相对分子质量单位 50ku处有表达条带;Western blot分析显示,rEm18 GST重组蛋白能被泡型棘球蚴病人阳性血清识别,具有良好的抗原性。 结论 成功构建了 pET41a Em18原核表达质粒,获得的 rEm18 GST重组蛋白具有生物活性和抗原性,有望应用于泡型包虫病诊断试剂的研制。
Objective To construct a prokaryotic expression plasmid for the E 18 gene of Echinococcus granulosus and obtain a highly expressed and bioactive Em18 recombinant protein, which lays the foundation for the development of diagnostic tools for echinococcosis. Methods Primers were designed by DNAman software. EcoR Ⅰ and Xho Ⅰ restriction sites were added to the 5 ’and 3’ ends of the primers respectively. The prokaryotic expression plasmids pMD18 T / Em18 were used as templates to amplify the Em18 gene fragment. The prokaryotic expression vector pET 41a (+) was constructed and the prokaryotic expression plasmid pET41a Em18 was constructed. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The correctness of its insertion sequence was confirmed by enzyme digestion, PCR and sequencing. The rEm18 GST recombinant protein and the GST recombinant protein were induced by IPTG, purified by glutathione Sepharose 4B affinity chromatography, and analyzed by SDS PAGE and Western blot. Results The sequencing showed that the prokaryotic expression plasmids of pET41a Em18 were correctly ligated and the insert of Em18 gene was 486 bp. SDS PAGE showed that the expression of Em18 gene was successfully expressed in the rEm18 GST recombinant protein and expressed in the relative molecular mass units of 50ku. Western blot analysis showed that rEm18 GST recombinant protein could be recognized by positive sera of hydatid cysts, Has good antigenicity. Conclusion The prokaryotic expression vector pET41a Em18 has been successfully constructed. The rEm18 GST recombinant protein has biological activity and antigenicity and is expected to be used in the development of diagnostic tools for.