人Troponin I的表达载体构建诱导表达和纯化及活性测定

以人肝脏cDNA文库为模板,通过PCR方法扩增出Tropon in基因,将其克隆到pCR-TOPO质粒中,构建表达质粒pET-22 b(+)-TnI,转化大肠杆菌BL 21(DE 3)-RPX并进行诱导表达,表达的目的蛋白占菌体总蛋白的20%以上并主要以可溶形式存在。建立了Tropon in I的非亲和色谱纯化工艺,纯化后蛋白产量约为20 mg/L,纯度在90%以上。体外活性实验表明重组蛋白具有良好的抗血管生成。

CONSTRUCTION EXPRESSION PURIFICATION AND ANTI-ANGIOGENESIS DETYERMINATION OF HUMAN TROPONIN I

The human troponin I gene was cloned from the human liver cDNA library by PCR amplification,and then sub-cloned into pCR-TOPO vector to construct expression plasmids pET-22 b(+)-Tnl.The pET-22 b(+)-Tnl plasmids were transformed in E.coli BL 21(DE 3)-RPX.Troponin I was expressed in E.coli upon IPDG induction.The target protein accounted for more than 20% of the total protein in a soluble form.The recombinant Tnl was purified by non-affinity chromatography.Troponin I yield was about 20 mg/L with a purity of over 90%.In vitro activity assay suggested that this recombinant has a good anti-angiogenesis activity.