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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。
OBJECTIVE: To clone Escherichia coli urease gene ureIABCEFGD from Streptococcus salivarius 57.I and test its relationship with the exogenous nickel ion concentration. Methods: The target gene was cloned into the first two and the second two were cloned, digested, ligated and sequenced. The ureIABCEFGD plasmid was transformed into competent E. coli TG-1, and different concentrations of NiCl2 were added respectively. After Nessler kit The amount of ammonia produced by the decomposition of urea was measured. The linear correlation analysis of ammonia production and NiCl2 concentration was carried out by SPSS17.0 software package. Results: The sequence of ureIABCEFGD was correct. After the clone was transformed into Escherichia coli, with the increase of exogenous NiCl2 concentration, the ammonia production of the recombinant strain increased rapidly (r = 0.9714, P <0.01) When the concentration was increased to 50μmol / L, the amount of ammonia production peaked and no longer increased with the concentration of NiCl2. CONCLUSION: The urease activity of ureIABCEFGD is positively correlated with exogenous nickel ions in a certain concentration range. This clone can be used to further study the regulation mechanism and method of urea decomposition activity.