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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。
Objective To construct a prokaryotic expression vector of aps gene of Streptococcus mutans and to express and purify the target protein, then to identify its biological function. Methods The genomic DNA of Streptococcus mutans UA159 was used as a template to amplify the coding region of aps gene of Streptococcus mutans and inserted into prokaryotic expression vector pASK-IBA43plus. The recombinant plasmid was transformed into E.coli Top 10 and induced by anhydrous tetracycline. The expression of His- After NTA purification, His-APS protein adenosine monophosphate sulfatase activity and ribonuclease activity were examined. Results The aps gene open reading frame was amplified by PCR. The aps gene prokaryotic expression vector pASK-aps was successfully constructed and expressed in E. coli Top 10. The molecular mass unit of the recombinant His-APS protein was 38ku , Which hydrolyzes pAp in the presence of Mg2 + and Mn2 + in a time- and concentration-dependent manner but does not degrade the Cy5-labeled random 6 nt oligonucleotide probe. Conclusion The recombinant His-APS protein possesses adenosine phosphate sulfatase activity in a time-and concentration-dependent manner, but it does not possess ribonuclease activity.