【摘要】 目的对新型进化免疫球蛋白(Ig)结合分子LD5进行原核表达和纯化,并鉴定其结合活性。方法将LD5基因片段克隆入原核表达载体pET32a(+),转化大肠杆菌后,IPTG诱导表达。以8mol/L尿素裂解菌体后,上清经Ni2+-NTA柱纯化,并对纯化蛋白进行SDS-PAGE及Western blot分析。以ELISA和Dot blot对LD5蛋白结合IgG、IgM活性进行鉴定。结果LD5分子在大肠杆菌中获得成功表达,表达的融合蛋白相对分子质量为59000。Western blot表明纯化的LD5蛋白能特异结合IgG。ELISA结果显示,LD5在结合IgM上较SpA具有明显的优势。结论LD5分子在大肠杆菌中已成功表达,纯化的LD5蛋白保留了天然Ig结合分子的结合活性。更多还原

【Abstract】 Objective To express evolved Ig-binding molecule LD5 in prokaryotic cells,purify the expressed product and determine its binding activity.Methods LD5 gene fragment was cloned into prokaryotic expression vector pET32a(+)and transformed to E.coli for expression under induction of IPTG.The recombinant E.coli bacteria was lysed with 8 mol/L urea,then LD5 protein was purified from the supernatant by Ni2+-NTA column chromatography,identified by SDS-PAGE and Western blot and determined for binding activities to IgG and IgM by ELISA and dot blot.Results LD5 fusion protein with a relative molecular mass of 59 000 was successfully expressed in E.coli.Western blot showed specific binding of the purified LD5 protein in IgG.ELISA proved that the binding activity of LD5 to IgM was significantly higher than that of SpA.Conclusion LD5 protein was successfully expressed in E.coli and showed similar binding activity to that of natural Ig binding molecules.更多还原