人DNase Ⅰ的表达、纯化及降解NETs活性研究

Expression,purification and NETs degradation activity of human DNase Ⅰ

为获得纯度较高的人脱氧核糖核酸酶Ⅰ(DNase Ⅰ)以研究其对中性粒细胞胞外诱捕网(neutrophil extracellulartraps,NETs)的降解作用,构建基因工程表达菌E. coli Rosetta (DE3)/p ET32a-His-DNase Ⅰ,乳糖诱导表达,经镍柱亲和纯化获得融合蛋白His-DNase Ⅰ。提取小鼠中性粒细胞,用佛波酯PMA刺激形成NETs,Sytox Green及荧光显微镜检测融合蛋白对NETs的降解活性。结果表明,成功构建人DNase Ⅰ基因克隆并在原核细胞中实现高效表达,纯化的His-DNase Ⅰ具备较高的核酸酶活性。本研究为进一步探究DNase Ⅰ的临床应用奠定了理论基础。

In order to study the effect of human deoxyribonuclease I (DNase I) on neutrophil extracellular traps (NETs) degradation,genetic engineering bacteria E.coli Rosetta (DE3)/pET32a-His-DNase I was constructed.The fusion protein His-DNase I was induced by lactose,and purified by Ni-affinity chromatography. Mouse neutrophils were extracted and stimulated with phorbol-12-myristate-13-acetate (PMA) to form NETs. The degradation activity of the fusion protein on NETs was detected by SytoxGreen and fluorescence microscopy. The results showed that the purified His-DNase I had high nuclease activity. This study provided the research foundation for further exploration of the clinical application of DNase I.