摘要
目的 克隆唾液链球菌57.Ⅰ的尿素酶基因,并在不添加外源性镍离子的情况下表达出有活性的尿素酶,以期为进一步研究口腔细菌的尿素分解机制和牙菌斑生物膜的产碱活性提供依据.方法 将尿素酶基因分成前、中、后三段,分别设计引物,应用聚合酶链反应（PCR）法进行克隆并测序鉴定;进而采用双酶切的方法 将分段克隆的产物逐步连接为完整的尿素酶基因;将含有完整尿素酶基因的质粒转化感受态大肠杆菌TG-1,经酚红脲酶试验检测其尿素分解活性.结果 所克隆的唾液链球菌57.Ⅰ尿素酶基因序列正确;无需添加氯化镍,该克隆能够在大肠杆菌中表达出有活性的尿素酶,分解培养基中的尿素产生氨,升高环境中的pH值.结论 本研究克隆的唾液链球菌尿素酶基因无需添加外源性镍离子就能够表达出尿素分解活性,可用于今后替代疗法防龋研究中构建更适合临床应用的产碱效应菌.
Objective To clone Streptococcus salivarius（Ss） 57. I urease gene, which can express ureolytic activity in Escherichia coli （Ec） without adding extra nickel ions. Methods Urease gene was cloned by polymerase chain reaction in three separate parts. The three separate plasmids were digested by specific restriction enzymes and ligated together. The expression of the complete urease gene in Ec was detected by phenol red assay and pH analysis. Results Urease gene of Ss 57. I was eventually cloned and proved correct. Urease activity of the obtained clone was positive in Ec. Without adding extra NiCl2, the recombinant Ec could hydrolyze urea to produce ammonia, resulting in the increase of pH value.Conclusions The clone of Ss urease gene obtained in this study could express ureolytic activity in Ec without adding extra nickel ions. The current clone can be used to construct ureolytic effector strain used in replacement therapy in caries prevention.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2010年第8期498-501,共4页
Chinese Journal of Stomatology
基金
国家自然科学基金（30572038）
上海市科学技术委员会科研计划（08ZR1416800、09DZ2272100）
上海市重点学科建设计划（S30206）
关键词
链球菌
口腔
尿素酶
镍
龋齿
Streptococcus,oralis
Urease
Nickel
Dental caries
