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目的原核表达金黄色葡萄球菌(Staphylococcus aureus,S.aureus)α-溶血素(α-hemolysin,Hla)及其突变体,并检测其免疫学活性。方法采用PCR法从S.aureus中扩增hla基因,将该基因克隆至原核表达载体p ET28a中,构建重组表达质粒p ET28a-hla,并利用点突变试剂盒进行突变,获得重组质粒p ET28a-hlaH35L。将两种重组表达质粒转化大肠埃希菌BL21(DE3),IPTG诱导表达;表达的重组蛋白经Ni-NTA亲和层析和CM离子交换层析纯化后,检测其溶血活性、免疫血清的抑制溶血活性及HlaH35L蛋白的免疫保护作用。结果重组表达质粒p ET28a-hla经双酶切和测序证明构建正确;重组质粒p ET28a-hlaH35L的测序结果显示,第35位氨基酸突变位点与设计相符。表达的Hla和HlaH35L蛋白相对分子质量约为36 000,均为可溶性表达,表达量约占菌体总蛋白的50%,纯化后纯度均在90%以上。Hla具有溶血活性,溶血比活为152 HU/mg;HlaH35L无溶血活性;抗Hla和抗HlaH35L血清均具有抑制Hla溶血的活性;在小鼠滴鼻攻击模型中,HlaH35L具有一定的保护作用。结论成功在大肠埃希菌中表达了具有良好免疫学活性的Hla及其突变体HlaH35L,为筛选S.aureus候选疫苗组分奠定了实验基础。
Objective To prokaryotically express Staphylococcus aureus (S. aureus) α-hemolysin (Hla) and its mutants, and to examine their immunological activity. Methods The hla gene was amplified from S. aureus by PCR and cloned into prokaryotic expression vector p ET28a. The recombinant plasmid p ET28a-hla was constructed and mutated by point mutation kit. The recombinant plasmid p ET28a- hlaH35L. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The expressed recombinant protein was purified by Ni-NTA affinity chromatography and CM ion exchange chromatography. The hemolysis activity and the inhibition of immune serum Hemolysis Activity and Immunoprotection of HlaH35L Protein. Results The recombinant plasmid pET28a-hla was confirmed by double enzyme digestion and sequencing. The sequencing result of the recombinant plasmid pET28a-hlaH35L showed that the amino acid mutation at position 35 was consistent with the design. The relative molecular mass of the expressed Hla and HlaH35L proteins was about 36 000, both of which were soluble, accounted for about 50% of the total bacterial proteins. The purity of the expressed Hla and HlaH35L proteins was above 90%. Hla has hemolytic activity with a specific hemolysis ratio of 152 HU / mg; HlaH35L has no hemolysis activity; both anti-Hla and anti-HlaH35L sera have anti-Hla hemolysis activity; HlaH35L has a protective effect in a mouse intranasal challenge model. Conclusion Hla and its mutant HlaH35L with good immunological activity were successfully expressed in Escherichia coli, which laid the experimental foundation for the screening of candidate vaccine components of S. aureus.