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葡萄球菌核酸酶的制备及性质研究

Studies on preparation and properties of Staphylococcus nuclease

  • 摘要: 为获得纯度较高的葡萄球菌核酸酶(SNase)以研究其与糖尿病的关系,构建基因工程表达菌E.coli BL21/pET28a-His-SNase,诱导其表达可溶性胞外蛋白,并对纯化的SNase进行初步性质研究。采用分子生物学方法设计和构建含有SNase基因的表达载体pET28a-His-SNase,再转入E.coli BL21(DE3)感受态细胞。经过乳糖诱导表达、超声破碎及镍柱亲和色谱等纯化步骤后,以SDS-PAGE和Western blot鉴定His-SNase,并对其性质进行了初步研究。结果表明,乳糖诱导后His-SNase能高效表达,亲和色谱后纯度高达85%以上,且其具备较高的核酸酶活性,作用pH范围较广,有很好的耐热性。本研究为进一步探究葡萄球菌核酸酶与糖尿病的关系奠定了基础。

     

    Abstract: In order to study the relationship between Staphylococcal nuclease(SNase)and diabetes mellitus, genetic engineering bacteria E. coli BL21/pET28a-His-SNase was constructed, the expression of soluble extracellular protein SNase was induced and the a preliminary research was made on it. An expression vector pET28a-His-SNase plasmid containing the His-SNase gene was constructed and transformed into E. coli BL21(DE3)competent cells. The protein was induced by lactose and purified by ultrasound destruction and Ni-affinity chromatography, respectively. It was then analyzed by SDS-PAGE and Western blot. The enzymatic properties for SNase has been preliminary studied as well. Results indicated that the purity of the correctly expressed fusion protein His-SNase was over 85%. SNase showed good activity within a wide range of pH and good heat resistance. This experiment might be a foundation work for the further study on the relationship between SNase and with diabetes.

     

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