金黄色葡萄球菌α-溶血素及其突变体的原核表达和免疫学活性

Prokaryotic expression and immunological activity of Staphylococcus aureus α-hemolysin and its mutant

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 express α-hemolysin of Staphylococcus aureus and its mutant,and determine the immunological activity of expressed products. Methods The hla gene was amplified by PCR from S. aureus and inserted into prokaryotic expression vector p ET28 a. The constructed recombinant plasmid p ET28a-hla was subjected to site-directed mutation to obtain recombinant plasmid p ET28a-hlaH35 L. The two recombinant plasmids were transformed to E. coli BL21（DE3） for expression under induction of IPTG. The expressed recombinant proteins were purified by Ni-NTA affinity chromatography and CM ion exchange chromatography,and determined for hemolytic activity,the inhibition of hemolytic activity of their antisera. Results The recombinant plasmids were constructed correctly as proved by restriction analysis and sequencing.Sequencing result showed that the mutation site at amino acid 35 was consistent with that designed. Recombinant Hla and HlaH35 Lproteins,each with a relative molecular mass of about 36 000,were expressed both in soluble forms,which contained about 50% of total somatic protein and reached a purity of more than 90% after purification. Hla showed hemolytic activity,of which the specific hemolytic activity was 152 HU / mg,while HlaH35 Lshowed no hemolytic activity.Both the antisera of recombinant Hla and HlaH35 Linhibited the hemolytic activity of Hla. HlaH35 Lshowed a certain protection against Staphylococcus aureus infection in mice. Conclusion Hla with good immunogenicity and its mutant HlaH35 Lwere successfully expressed in E. coli,which provided an experimental basis for screening of a candidate antigen of S. aureus vaccine.