【摘要】 灵杆菌(Serratia marcescens)核酸酶高效的核酸去除能力与较强的稳定性使其在生物医药领域有广泛的应用,而如何高效去除反应体系中添加的微量核酸酶成为了重要的生产问题。为建立反应后核酸酶的快速去除系统,本研究构建了融合Strep-tagⅡ标签的灵杆菌非特异核酸酶(Strep-tagⅡfused nonspecific nuclease,SNU),并对融合蛋白的催化特性以及去除效果进行分析。依据灵杆菌核酸酶基因序列,设计融合Strep-tagⅡ编码序列的引物克隆目标基因并构建融合表达载体p LLP-Omp A+snu,并将测序正确的重组质粒转化宿主菌大肠杆菌(Escherichia coli)BL21并进行诱导表达。融合Strep-tagⅡ的核酸酶以包涵体形式过量表达,通过优化包涵体纯化条件获得高纯度包涵体。经包涵体复性可获得高活性的融合蛋白,复性率大于60%。复性蛋白经二乙氨基乙基(diethylaminoethyl,DEAE)-琼脂糖凝胶(sepharose)阴离子交换层析进一步纯化获得了高纯度的融合蛋白。融合蛋白的纯化效率可以达到18.695 mg/L。活性检测表明该融合蛋白能够高效降解DNA与RNA,其最适温度为37℃,最适p H为8.0,比活力达到1.53×105U/mg。500 mmol/L以下尿素对酶活性几乎没有影响,而高于50 mmol/L Mg Cl2、大于2 mmol/L乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)则会对活性产生一定的抑制作用。不同二价金属离子对酶活性影响的分析表明,Mg2+和Mn2+对重组核酸酶活性有明显的促进作用,Zn2+、Cu2+和Ca2+则没有促进作用,而Co2+、Ni2+和Fe2+对重组核酸酶的活性促进不显著。通过链霉亲和素(Streptavidin)-Sepharose CL6B能够实现融合Strep-tagⅡ标签的核酸酶的高效去除。融合Strep-tagⅡ标签的灵杆菌非特异核酸酶的重组表达、包涵体纯化-复性体系以及去除系统的建立为灵杆菌非特异核酸酶的实践应用提供了基础资料。更多还原

【Abstract】 Serratia marcescens nuclease has been widely used in the biomedicine field due to its high efficiency of nucleic acid removal and its strong stability.However,the arising problem is how to efficiently remove the trace amount nuclease left in the reaction system.In order to establish the quick removal system for the nuclease residue,S.marcescens nuclease fused with Strep-tagⅡ was designed and the characteristics of the recombinant nuclease was analyzed.The primers were designed based on the sequence of S.marcescensnuclease published in Gen Bank and the sequence of Strep-tag Ⅱ was fused into the forward primer.The nuclease gene was amplified by PCR and ligated into expression vector p LLP-Omp A.The verified recombinant vector was transformed into Escherichia coli BL21 and induced by isopropyl-β-d-thiogalactoside(IPTG),and the recombinant nuclease was overexpressed in the form of inclusion body.The purified inclusion body was dissolved in 8 M urea and renatured by gradient dialysis.The purity of renatured protein from inclusion body was approximate 60%.The renatured nuclease was further purified through DEAE-Sepharose and the final yield of the fusion protein was 18.695 mg/L.Catalytic characteristics analysis results showed optimal temperature of recombinant nuclease was 37 ℃ and optimal p H was 8.0 and its specific activity was1.53×105U/mg.The catalytic activity was not inhibited by urea of 500 mmol/L while was inhibited by over 2mmol/L ethylene diamine tetraacetic acid(EDTA).Mg2 +was necessary for the catalytic activity of the nuclease while inhibited by over 50 mmol/L Mg Cl2.Evaluation the effects of different bivalent metallic cations on catalytic activities showed that the activity was strongly enhanced by Mn2 +,Mg2 +and lightly enhanced by Co2 +,Ni2 +and Fe2 +while not by Zn2 +,Cu2 +and Ca2 +.The recombinant nuclease could be efficiently removed by Streptavidin-Sepharose CL 6B resin.The established overexpression of Strep-tag Ⅱtagged S.marcescens nuclease and removal system will provide the foundation for the practice application of S.marcescens nuclease.Ⅱ更多还原