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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 prokaryotic express and purify Listeriolysin O (LLO). Methods The gene of LLO hly was amplified by PCR and inserted into pET-32a vector. The recombinant plasmid pET-hly was constructed and transformed into E. coli BL21 (DE3). The recombinant protein was induced by IPTG. The recombinant protein was purified by gel electrophoresis and analyzed by Western blot. Results The cloned hly gene was 1 515 bp in length, which shared 99% homology with the hly gene in GenBank. The recombinant plasmid pET-hly was identified by restriction enzyme digestion and constructed correctly. The relative molecular mass of the expressed recombinant protein Was 55 000, accounting for 45.2% of the total bacterial protein. The purified recombinant protein reacted with positive Listeria monocytogenes. Conclusion The prokaryotic expression and purification of LLO have been successfully established, which lays the foundation for the further development of diagnostic kit.