单增李斯特菌核糖核酸酶Rnase Ⅲ RncS氨基酸突变对其降解RNA活性的影响

Effects of Amino Acid Mutation in the Rnase Ⅲ RncS of Listeria monocytogenes on RNA Degradation Activity

为研究单增李斯特菌(LM)核糖核酸酶Rnase Ⅲ RncS氨基酸突变对RNA降解活性的影响。利用生物信息学软件分析单核细胞增生李斯特菌(LM)野毒株SB5中rncS基因编码的Rnase Ⅲ的结构域,并选择关键氨基酸利用基因重叠延伸PCR(SOE-PCR)技术对其进行了基因突变;然后将rncS突变基因片段D50A、E122A克隆至表达载体pET-32a(+),在大肠杆菌中利用IPTG进行诱导表达;应用SDS-PAGE和Western Blot鉴定重组蛋白的表达情况及其抗原特异性;通过体外酶活试验研究其对RNA降解活性的影响。结构域分析结果显示,LM-Rnase Ⅲ氨基酸序列含有1个双链RNA结合结构域(DSRM)和1个核酸酶结构域(RIBOc),其中结构域RIBOc含有5个活性位点。SDS-PAGE检测结果显示,表达的重组突变型Rnase Ⅲ -D50A和Rnase Ⅲ -E122 A蛋白相对分子质量均为42.5 kD,与理论值相符;Western blot分析表明重组突变型Rnase Ⅲ -D50A和Rnase Ⅲ -E122A蛋白可与LM阳性血清发生免疫学反应。体外酶活实验表明,Rnase Ⅲ发挥降解活性依赖于Mn2+或Mg2+,将其第50位天冬氨酸突变后,Rnase Ⅲ RncS的降解活性有所降低(P<0.001);第122位谷氨酸突变后,Rnase Ⅲ RncS降解活性极显著下降(P<0.0001),提示第122位谷氨酸是维持LM Rnase Ⅲ RncS酶活性的关键位点。

To investigate the effect of amino acid mutation in Listeria monocytogenes(LM)ribonuclease RnaseⅢRncS on RNA degradation activity,bioinformatics software was used to analyze the domains of RnaseⅢencoded by rncS gene of LM wild-type strain SB5,and gene overlap extension PCR(SOE-PCR)was to perform its gene mutations.Then the rncS mutant gene fragments D50 A and E122 A were cloned into the expression vector pET-32 a(+),and induced by IPTG in Escherichia coli to generate mutant RnaseⅢ.SDS-PAGE and Western Blot were adapted to detect the expression form and the antigen specificity of fusion protein,respectively.Finally,the enzyme activity assay in vitro was to determine the effect of amino acid mutation on RNA degradation activity.The domain analysis revealed that the LMRnaseⅢprotein amino acid sequence included a nuclease domain(RIBOc),in which there were 5 active sites,and a double-stranded RNA binding domain(DSRM).The results of SDS-PAGE showed that the relative molecular masses of the recombinant mutants RnaseⅢ-D50 A and RnaseⅢ-E122 A were 42.5 k D,which was consistent with the theoretical values.Western Blot showed that the recombinant mutant RnaseⅢD50 A and RnaseⅢ-E122 A reacted with positive serum against LM.Enzyme activity experiments demonstrated that the degradation activity of RnaseⅢwas dependent on Mn2+or Mg2+.After the 50 th aspartic acid was mutated,the degradation activity of RnaseⅢRncS reduced(P<0.001),while it significantly(P<0.0001)decreased after the mutation of the 122 nd glutamate,suggesting that the 122 nd glutamate was the key site for maintaining the activity of LM RnaseⅢ.This study provides a scientific basis for further revealing the characteristics and mechanism of LM RncS.