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

摘要: 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 extracellular traps，NETs)的降解作用，构建基因工程表达菌E.coli Rosetta(DE3)/pET32a-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，SytoxGreen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。

Abstract: 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.