Directional evolution of TetR protein and development of a fluoroimmunoassay for screening of tetracyclines in egg
Directional evolution of TetR protein and development of a fluoroimmunoassay for screening of tetracyclines in egg
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TetR蛋白的定向进化和鸡蛋中四环素筛选荧光免疫分析的发展
DOI：
10.1016/j.microc.2019.104184
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发表时间：
2019-11-01
影响因子：
4.8
通讯作者：
Liu, Jing
中科院分区：
文献类型：
--
作者：
Wang, Ge;Xia, Wan Qiu;Liu, Jing
本研究将TetR蛋白结合口袋中的接触氨基酸Pro105直接用Phe和Tyr取代，分别产生突变体1和突变体2。两个突变体同时识别9种四环素药物，并且与亲本TetR相比，它们对9种药物的亲和力显着增加，敏感性增加1.5-13.3倍。分子对接显示，稳定性提高的突变体2比结合能大幅增加的突变体1对9种药物表现出更高的敏感性。然后将突变体2与荧光标记示踪剂结合，开发出一种一步式竞争性荧光免疫分析方法，用于同时筛选鸡蛋中的9种药物。将示踪剂和样品提取物添加到突变体2包被的微孔板孔中进行竞争，并直接激发荧光强度以进行分析物定量。结果表明，9种药物的半数抑制浓度范围为3.1-17.2 ng/mL，检出限范围为0.3-5.8 ng/mL。因此，该方法可作为鸡蛋中9种四环素类药物残留监测的常规筛查工具。
In this study, the contact amino acid Pro105 in the binding pocket of TetR protein was directly substituted with Phe and Tyr to produce the mutant1 and the mutant2 respectively. The two mutants simultaneously recognized 9 tetracycline drugs, and they showed highly increased affinities to the 9 drugs with 1.5-13.3 folds of increased sensitivity in comparison with the parental TetR. The molecular docking revealed that the mutant2 with improved stability showed higher sensitivity to the 9 drugs than the mutant1 with highly increased binding energy. Then the mutant2 was combined with a fluorescent-labeled tracer to develop a one-step competitive fluoroimmunoassay for simultaneous screening of the 9 drugs in egg. The tracer and the sample extract were added into the mutant2-coated microplate wells for competition, and the fluorescence intensity was excited directly for analyte quantification. Results showed that the half of inhibition concentrations for the 9 drugs were in the range of 3.1-17.2 ng/mL, and the limits of detection were in the range of 0.3-5.8 ng/mL. Therefore, this method could be used as a routine screening tool to monitor the residues of the 9 tetracyclines in egg.