CAJ | 학술논문

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.
목적 극륭타액련구균57.Ⅰ적뇨소매기인,병재불첨가외원성얼리자적정황하표체출유활성적뇨소매,이기위진일보연구구강세균적뇨소분해궤제화아균반생물막적산감활성제공의거.방법 장뇨소매기인분성전、중、후삼단,분별설계인물,응용취합매련반응(PCR)법진행극륭병측서감정;진이채용쌍매절적방법 장분단극륭적산물축보련접위완정적뇨소매기인;장함유완정뇨소매기인적질립전화감수태대장간균TG-1,경분홍뇨매시험검측기뇨소분해활성.결과 소극륭적타액련구균57.Ⅰ뇨소매기인서렬정학;무수첨가록화얼,해극륭능구재대장간균중표체출유활성적뇨소매,분해배양기중적뇨소산생안,승고배경중적pH치.결론 본연구극륭적타액련구균뇨소매기인무수첨가외원성얼리자취능구표체출뇨소분해활성,가용우금후체대요법방우연구중구건경괄합림상응용적산감효응균.
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.