单增李斯特菌溶血素O的原核表达及纯化

Prokaryotic expression and purification of Listeria monocytogenes Listeriolysin O

目的原核表达并纯化单增李斯特菌溶血素O(Listeriolysin O,LLO)。方法 PCR扩增LLO hly基因,并插入pET-32a载体,构建重组表达质粒pET-hly,转化入大肠杆菌BL21(DE3),IPTG诱导表达,切胶纯化重组蛋白,并进行Westernblot分析。结果克隆的hly基因长1 515 bp,与GenBank中登录的hly基因的核苷酸序列同源性为99%;重组表达质粒pET-hly经酶切鉴定构建正确;表达的重组蛋白相对分子质量约为55 000,表达量占细菌总蛋白的45.2%;纯化的重组蛋白可与单增李斯特菌阳性血清反应。结论已成功原核表达并纯化了LLO,为下一步诊断试剂盒的研制奠定了基础。

Objective To express Listeria monocytogenes Listeriolysin O（LLO）in prokaryotic cells and purify the expressed product.Methods The hly gene of LLO was amplified by PCR and inserted into vector pET-32a.The constructed recombinant plasmid pET-hly was transformed to E.coli BL21（DE3）and induced with IPTG.The expressed recombinant protein was purified by cutting the gel and analyzed by Western blot.Results The hly gene at a length of 1 515 bp was cloned,of which the homology was 99% to that reported in GenBank.Restriction analysis showed that recombinant plasmid pET-hly was constructed correctly.The expressed product,with a relative molecular mass of about 55 000,contained 45.2% of total somatic protein.The purified recombinant protein showed specific reaction with Lm positive sera.Conclusion LLO was successfully expressed in prokaryotic cells and purified,which laid a foundation of preparation of diagnostic kit.