摘要
目的:克隆唾液链球菌57.Ⅰ的尿素酶基因ureIABCEFGD转化大肠杆菌,检测重组菌株的尿素分解活性与外源性镍离子浓度的关系。方法:将目的基因分成前、后2段分别克隆,再酶切、连接并测序鉴定,得到的尿素酶基因ureIABCEFGD质粒转化感受态大肠杆菌TG-1,分别添加不同浓度的NiCl2,经Nessler试剂盒检测其分解尿素的产氨量,采用SPSS17.0软件包对产氨量和NiCl2浓度进行线性相关分析。结果:克隆的尿素酶基因ureIABCEFGD序列正确,该克隆转化大肠杆菌后,随着外源性NiCl2浓度增高,重组菌株的产氨量迅速增高,呈正相关(r=0.9714,P<0.01);当NiCl2浓度增加到50μmol/L时,产氨量趋于峰值,不再随NiCl2的浓度而增加。结论:唾液链球菌尿素酶基因ureIABCEFGD尿素分解活性的表达在一定浓度范围内与外源性镍离子的添加呈正相关,该克隆可用于进一步研究尿素分解活性的调控机制和方法。
PURPOSE： To obtain the clone of Streptococcus salivarius 57. I urease genes urelABCEFGD and investigate the relationship between ureolytic activity expression of this clone in Escherichia coli and nickel ions. METHODS： The target gene was cloned by polymerase chain reaction in 2 parts separately. Then, 2 plasmids were digested by specific restriction enzymes and ligated together. The obtained plasmids were subjected to nucleotide sequence analysis and transformed into E.coli TG-1. The recombinant E.coli was added without or with different level of NiC12. The amount of ammonia generated by ureolytic activity of each sample was measured by Nessler＇s assay. SPSS 17.0 software package was used for correlation analysis. RESULTS： The clone of urease genes urelABCEFGD was proved by sequence analysis and BLAST search. The amount of ammonia generated by the recombinant strain had a positive correlation with the level of NiCl2 （r=0.9714,P〈0.01）. When the level of NiC12 was 50 μmol/L, the amount of ammonia reached maximum and would have little variance despite the increase of NiC12 level. CONCLUSIONS： Ureolytic activity expression of urelABCEFGD has a positive correlation with the level of added NiC12 not exceeding 50 μmol/L. The present clone can be used to further investigate the regulation of the ureolytic activity expression of Streptococcus salivarius urease gene. Supported by Natural Science Foundation of Shanghai Municipality（O8ZR1416800） and Research Fund of Science and Technology Committee of Shanghai Municipality（11411950900）.
出处
《上海口腔医学》
CAS
CSCD
北大核心
2013年第5期504-507,共4页
Shanghai Journal of Stomatology
基金
上海市自然科学基金(08ZR1416800)
上海市科学技术委员会资助项目(11411950900)~~
关键词
链球菌
尿素酶
镍
龋病
Streptococcus
Urease
Nickel
Dental caries
