心肌肌钙蛋白Ⅰ的融合表达和分离纯化

Fusion expression in E.coli and affinity purification of troponin Ⅰ

目的研究人心肌肌钙蛋白(cTnⅠ)在大肠杆菌中稳定高效的融合表达和分离纯化,以满足临床诊断的需要。方法人cTnⅠDNA序列由pdf 5-cTnⅠ质粒经PCR亚克隆而得,然后将其插入pET32a(+)载体,构建成pET32a(+)-cTnⅠ表达质粒,以大肠杆菌BL21(DE3)为表达菌,在IPTG诱导下进行表达。结果硫氧还蛋白(TrxA)-cTnⅠ融合蛋白得到高效表达,并经金属螯合亲和色谱一步纯化得到目的蛋白质。试剂盒检测结果表明该重组蛋白质具有cTnⅠ免疫原活性。结论构建了pET32a(+)-cTnⅠ重组质粒,并在大肠杆菌中获得高效表达,为建立急性心肌梗死早期诊断试剂奠定了基础。

Purpose The present work was to express a stable heterologous human cTnⅠ fusion protein to TrxA as part of an overall strategy for the isolation and purification of enough material for use as a diagnostic calibrator. Methods The DNA sequence was isolated from the cDNA plasmid pdf 5-cTnⅠ by PCR and cloned as a fusion to the C-termius of TrxA and the recombinant cTnⅠ expression vector was constructed and expressed in host E. coli BL21（DE3）. Results When induced with IPTG,the pET32a（ ＋ ）-cTnⅠ was highly expressed in E. coli. The fusion protein was affinity-purified over a Histrap Column. The troponinⅠ fusion protein was assayed and found to exhibit similar immunogenic response relative to natural cardiac troponinⅠ, Conclusion The results demonstrated that TrxA-cTnⅠ was successfully expressed. The recombinant cTnⅠ was applicable in early detection of acute myocardial infarction.