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目的原核表达并纯化单核李斯特菌(Listeria monocytogenes,LM)溶血素(Listeriolysin O,LLO)蛋白。方法用1对LM LLO基因特异性引物从LM基因组DNA中扩增LLO基因,并构建重组原核表达质粒pET-30a(+)/rLLO,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的rLLO蛋白经镍离子金属螯合柱纯化后,进行SDS-PAGE及质谱分析。结果重组表达质粒pET-30a(+)/rLLO经PCR、双酶切及测序,证明构建正确。表达的rLLO融合蛋白相对分子质量约为78 000,主要以包涵体形式表达。质谱分析显示,两个相对分子质量分别为35 000和45 000的小分子也均为LLO蛋白。结论已成功地在大肠杆菌中表达了rLLO蛋白,并进行了纯化,为进一步研究LLO蛋白的生物学功能及研制基于LLO蛋白的特异性诊断制剂奠定了基础。
Objective To express and purify Listeria monocytogenes (LLO) protein in prokaryotic cells. Methods LLO gene was amplified from LM genomic DNA using a pair of LM LLO gene-specific primers and the recombinant prokaryotic expression plasmid pET-30a (+) / rLLO was constructed and transformed into E. coli BL21 (DE3) for expression under IPTG. The expressed rLLO protein was purified by nickel ion metal chelate column and analyzed by SDS-PAGE and mass spectrometry. Results The recombinant plasmid pET-30a (+) / rLLO was confirmed by PCR, double enzyme digestion and sequencing. The expressed rLLO fusion protein has a relative molecular mass of about 78 000 and is mainly expressed in inclusion bodies. Mass spectrometry analysis showed that two small molecules with relative molecular weights of 35 000 and 45 000 respectively were also LLO proteins. Conclusion The rLLO protein was successfully expressed in Escherichia coli and purified, which laid the foundation for the further study of the biological function of LLO protein and the development of a specific diagnostic agent based on LLO protein.