中文摘要：

目的构建变异链球菌aps基因原核表达载体,表达纯化目的蛋白,继而对其生物学功能进行初步鉴定。方法以变异链球菌UA159株基因组DNA为模板PCR扩增变异链球菌aps基因编码区,插入原核表达载体pASK-IBA43plus,转化E.coli Top 10,无水四环素诱导His-APS蛋白表达,经Ni-NTA纯化后,检测His-APS蛋白腺苷磷酸硫酸酶活性及核糖核酸酶活性。结果 PCR扩增出933bp的aps基因开放读码框,成功构建aps基因原核表达载体pASK-aps,并在E.coli Top 10中诱导表达,SDS-PAGE检测重组His-APS蛋白分子质量单位为38ku,该蛋白在Mg2＋及Mn2＋存在条件下能水解pAp,且呈时间及浓度依赖性,但不能降解Cy5标记的随机6nt寡核苷酸探针。结论重组His-APS蛋白具有腺苷磷酸硫酸酶活性,呈时间及浓度依赖性,但不具有核糖核酸酶活性。

英文摘要：

Objectives To construct a prokaryotic expression plasmid containing the aps gene of Streptococcus mutans,express this gene in E.scherichia coli,and then identify the function of the protein after purification.Methods Genomic DNA of S.mutans US159 was used as template to amplify the open reading frame of the aps gene using PCR.The PCR fragment was then inserted into pASK-IBA43 plus after digestion with EcoRⅠ/BamHⅠ to construct the prokaryotic expression plasmid pASK-aps.E.coli Top10 with the plasmid pASK-aps was then incubated in LB media containing 100 mg/ml ampicillin.His-tagged APS protein was purified with Ni-NTA after induction with anhydrotetracycline.The pAp phosphatase and RNase activity of His-tagged APS was then identified.Results PCR amplification yielded an aps gene with an open reading frame of 933 bp and the fragment was then inserted into pASK-IBA43 plus via EcoRⅠ/BamHⅠ.The sequence of the aps gene fragment proved to be correct according to sequencing.The results of SDS-PAGE demonstrated that the recombinant His-tagged APS protein had a molecular weight of about 38 ku.This His-tagged APS protein can convert pAp to AMP and inorganic phosphate in vitro in the presence of Mg2＋ or Mn2＋,but it cannot degrade Cy5-tagged RNA oligos even in the presence of high concentrations of Mg2＋ or Mn2＋.Conclusion S.mutans APS yielded time and dose-dependent pAp phosphatase activity in vitro.This study provides grounds for the further investigation of the function of the S.mutans aps gene.