基于调控DNA序列变化的MphR(A)蛋白类ELISA系统性能提升研究

Study on performance improvement of ELISA-type systems based on MphR(A) protein via DNA sequence regulation

中文关键词:  大环内酯类抗生素  类ELISA系统  MphR(A)蛋白

英文关键词:macrolide antibiotics  ELISA-type system  MphR(A) protein

中文摘要:

      目的 考察DNA序列对利用MphR(A)蛋白和启动子mph(A)序列的结合-解离特性构建的大环内酯类抗生素的体外类酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)检测系统性能的影响。方法 通过MphR(A)蛋白与3段野生型DNA序列及其他突变序列构建类ELISA检测系统, 先通过添加和不添加红霉素作用, 获得各DNA所得的最大和最小OD450, 以最大与最小OD450值相差较大、最小OD450值接近0作为表现较好的标准, 再对表现较好的系统计算红霉素反应的半数抑制浓度(median inhibitory concentration, IC50)值。结果 MphR(A)蛋白可以和不同DNA序列结合, 从而获得不同的检测性能。采用突变序列AC4-DNA建立的类ELISA系统IC50值为(2.33±0.70) ng/mL, 低于野生型DNA所得的IC50 [(3.95±1.18) ng/mL], 系统灵敏度提升。结论 通过DNA调控该类ELISA系统是一种可行的手段。此外, 在野生型A-DNA、B-DNA、C-DNA均发现一段22 bp伪回文结构, 该伪回文结构符合Tet蛋白家族的DNA序列特征, 认为是该伪回文结构是与蛋白结合的核心序列。

英文摘要:

      Objective To investigate the effects of DNA sequences on the performance of the in vitro ELISA-type detection system for macrolide antibiotics constructed based on the binding-dissociation characteristics of the MphR(A) protein with the promoter mph(A) sequence. Methods An ELISA-type detection system was established using MphR(A) protein with 3 wild DNA sequences andmutated sequences, the maximum and minimum OD450 values corresponding to DNAs were obtained by with and without erythromycin, taking the larger differences between the maximum and minimum OD450 values and the minimum OD450 value close to 0 as good performance criteria, and the median inhibitory concentration (IC50) value of the ELISA-type system with better performance for erythromycin was calculated. Results MphR(A) protein could bind to some different DNA sequences so that obtain different detection performance. The IC50 of the ELISA-type system established using AC4-DNA was (2.33±0.70) ng/mL, which was lower than the IC50 [(3.95±1.18) ng/mL] using wild-type DNA. Conclusion It is a feasible method to improve performances of the ELISA-type system via DNA regulation. In addition, a 22 bp pseudo-palindromic structure is found in wild-type A-DNA, B-DNA, and C-DNA. The pseudo-palindromic structure conforms to the DNA sequence characteristics of the Tet protein family, and considered is the core sequencebinding to MphR(A) protein.