人工表达β2肾上腺素受体的β激动剂多残留检测

Mutiresidue Detection of β Aeceptor Agonists in β2Adrenergic Receptor by Artificial Expression

摘要:

为提高人工表达的β2肾上腺素受体（β2adrenergic receptor，β2AR）蛋白的活性并对其进行活性鉴定，利用分子对接技术将β2AR与15种β受体激动剂进行柔性分子对接，依据筛选出的与β受体激动剂结合力较好的β 2AR分子结构，人工合成和改造β2AR基因，构建重组表达质粒转入大肠杆菌BL21(DE3)，表达出具有活性的受体蛋白。SDS-PAGE鉴定结果显示，受体蛋白大小在47 ku左右。活性鉴定结果显示，与克伦特罗、莱克多巴胺及非诺特罗均能够特异性结合，OD值分别为0.45、0.32、0.36，受体蛋白与激动剂的结合力与其活性鉴定结果基本一致。标准检测曲线结果显示，受体蛋白对β受体激动剂类药物具有一定检测能力。利用分子对接技术，成功获得一定活性的β2AR受体蛋白，为β2AR在β受体激动剂多残留检测中的应用奠定了基础。

Abstract:

This study was directed to improve the activity of β2 adrenergic receptor and to identify the activity ofβ2 adrenergic receptor by artificial expression.We used molecular docking technology to separately model the interaction between β2adrenergic receptor and fifteen kinds ofβ receptor agonists. Based on the molecular structure of β2 adrenergic receptor, the β receptor agonists with best binding in molecular docking was selected, and the β2adrenergic receptor gene was synthesized and optimized. The recombinant expression plasmid was constructed to be transferred to Escherichia coil BL21(DE3), which expressed the active receptor protein successfully. SDS-PAGE of recombinant expression plasmid showed that theβ2 adrenergicreceptor protein was approximately 47 ku. The results of the receptor activity showed that the receptor protein was capable of specific reaction with Clenbuterol, ractopamine, and fenoterol respectively. OD values were 0.45, 0.32 and 0.36 respectively, and the binding ability of the receptor protein and β receptor agonists was consistent with the results of the activity identification basically. Testing curve shows that β2 adrenergic receptor protein have the ability to detect β receptor agonists. Using molecular docking technology, we successfully obtained the activeβ2adrenergic receptor protein, which laid the foundation for the application of β2adrenergic receptor to the multresidue detection of β receptor agonists.