磷酸腺苷-磷酸酶活性参与变异链球菌抗氧化应激反应.DOC

PAGE PAGE 6 变异链球菌腺苷磷酸硫酸酶的原核表达及功能研究 张加勤，侯香华**，宋秀宇** （厦门大学附属第一医院检验科，厦门，361003，中国） 【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-IBA43 plus，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诱导表达，SDS检测重组His-APS蛋白分子量为38KDa，该蛋白在Mg2+及Mn2+存在的条件下能水解pAp，且呈时间及浓度依赖性，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，不具有核糖核酸酶活性，为进一步研究aps基因功能提供依据。 【关键字】变异链球菌；腺苷磷酸硫酸酶；核糖核酸酶 Study on the Purification and bio-functions OF pAp Phosphatase of Streptococcus mutans * ZHANG Jia-qin, HOU Xiang-hua**, SONG Xiu-Yu** Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen Xiamen 361003 , China 【Abstract】 Objective　To construct a prokaryotic expression plasmid containing the aps gene of Streptococcus mutans，express this gene in E. 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 aps gene. PCR fragment was inserted into pASK-IBA43 plus after digesting by EcoRI /BamHI to construct the prokaryotic expression plasmid. The His-tagged APS protein was purified by Ni-NTA after being induced by anhydrotetracycline in LB media contain 100mg/mL Ampicillin. Then pAp Phosphatase and RNase activity of His-tagged APS were identified. Results PCR amplification yield a 933bp open reading frame of aps gene and was inserted into pASK-IBA43 plus via EcoRI /BamHI. The sequence of the 933bp fragment proved to be correct by sequencing. The results of SDSdemonstrateded that the molecular weight of the recombinant protein was about *【基金项目】国家自然科学基金（No.；福建省自然科学基金（No. 2010D018）；福建省卫生厅青年基金（No.2010-2-90）。 **【通讯作者】宋秀宇，E-mail: songxyxm@,侯香华，E-mail: xianghuahou@ 【作者简介】张加勤（1980-），男，博士，研究方向：变异链球菌应激耐受及表达调控，E-mail：jqzhangxm@。 38KD. This His-tagged APS protein can converts pAp to AMP and inorganic phosphate in vi