人溶菌酶基因的原核表达及其生物学活性

目的在大肠杆菌中表达人溶菌酶(hLYZ)基因,并检测其生物学活性。方法将人溶菌酶基因克隆至带有GST融合标签的原核表达载体pGEX-4T-1上,转化大肠杆菌BL21(DE3),经IPTG诱导表达。表达产物经亲和层析纯化后,进行Western blot鉴定,并用溶壁微球菌比浊法检测酶活性,热激法检测其生物学活性。结果经PCR、双酶切鉴定和核苷酸序列测定,表明重组表达质粒pGEX-4T-hLYZ构建正确。SDS-PAGE显示,在相对分子质量约40000处可见目的蛋白条带,表达量占菌体总蛋白的46.5%,目的蛋白在裂解沉淀中占17.0%,在裂解上清中占58.3%,主要以可溶形式表达。纯化后,重组蛋白纯度可达95%以上,且具有良好的反应原性。重组hLYZ酶活性为2389U/mg,对金黄葡萄球菌和大肠杆菌具有抑制作用。结论已成功地在大肠杆菌中表达了可溶性重组人溶菌酶,且具有较高的生物学活性。

Prokaryotic Expression and Biological Activity of Human Lysozyme

Objective To express human lysozyme(hLYZ)gene in E.coli and determine the biological activity of expressed product.Methods hLYZ gene was cloned into prokaryotic expression vector pGEX-4T-1 with GST fusion labeling, and the constructed recombinant plasmid was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by affinity chromatography and identified by Western blot, then determined for enzyme activity by Micrococcus lysodeikticus turbidimetric method and for biological activity by heat shock method.Results PCR, restriction analysis and nucleotide sequencing proved that recombinant expression vector pGEX-4T-hLYZ was constructed correctly.SDS-PAGE showed a target protein band with relative molecular mass of about 40 000.The expressed product contained 46.5% of total somatic protein and mainly existed in a soluble form, accounting 17.0% of precipitate and 58.3% of supernatant of lysed recombinant E.coli.After purification, the expressed protein reached a purity of more than 95% and showed good reactogenicity.With an enzyme activity of 2 389 U / mg, the recombinant hLYZ showed inhibitory effect on E.coli and Staphylococcus aureus.Conclusion Soluble hLYZ was successfully expressed in E.coli and showed high biological activity.