猪链球菌2型核糖核酸酶Ⅲ的克隆表达及催化特性分析

Cloning,expression and catalytic properties of ribonucleaseⅢfrom Streptococcus suis serotype 2

为了研究猪链球菌2型(SS2)核糖核酸酶Ⅲ(RNaseⅢ)的催化特性,利用大肠杆菌BL21(DE3)异源表达猪链球菌2型RNaseⅢ(SS-RNaseⅢ)并通过镍柱亲和层析的方法分离纯化。异源表达研究显示,重组蛋白RNaseⅢ大部分为可溶性表达;催化特性研究显示,RNaseⅢ主要依赖Mg^(2+)或Mn^(2+)发挥催化功能,并在不同离子浓度和pH值环境下具有不同的催化活性;底物特异性研究显示,RNaseⅢ特异性切割长度为30 bp的双链RNA,同时对内源性的rnc mRNA和5S rRNA具有良好的特异性。上述结果表明,相较于其他细菌RNaseⅢ,SS-RNaseⅢ具有独特的催化特性,为深入理解RNaseⅢ如何介导SS2致病机制奠定了基础。

In order to identify the catalytic properties of RNaseⅢStreptococcus suis serotype 2 RNaseⅢ(SS-RNaseⅢ),we used Escherichia coli BL21(DE3)to heterologously express RNaseⅢof Streptococcus suis serotype 2,and the bacterial RNaseⅢwas then isolated and purified by nickel column affinity chromatography.Next,various catalytic properties of SS-RNaseⅢwere analyzed.The results were that heterologous expression showed most of the recombinant protein RNaseⅢto be soluble,that the catalytic properties showed RNaseⅢmainly relying on Mg^(2+)or Mn^(2+)to perform catalytic functions with different catalytic activities under different ion concentrations and pH environments,that substrate specific analysis showed RNaseⅢto specifically cleave double stranded RNA with a length of 30bp,with good specificity for endogenous rnc mRNA and 5S rRNA.The above results indicated that SS-RNaseⅢhad unique catalytic properties,which provided theoretical basis for a deeper understanding of how SS-RNaseⅢmediates the pathogenesis of Streptococcus suis serotype 2.