摘要
依据大肠杆菌密码子偏好性,设计合成β_2肾上腺素受体(β_2 adrenergic receptor,β_2AR)基因序列,应用大肠杆菌无细胞系统对其进行高效表达,经负载镍离子的顺磁颗粒Magne His?Ni-Particles纯化后,对受体蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和活性鉴定。结果表明:改造后的β_2AR基因密码子适应指数(codon adaptation index,CAI)为0.96,GC含量从58%降低到46.17%,更有利于该基因在大肠杆菌系统中的表达。表达体系中优化后的Mg^(2+)浓度为22 mmol/L,此时表达量为1 250μg/mL。SDS-PAGE分析显示,纯化蛋白在47 k Da左右出现清晰的特异性条带,与预期结果一致,纯度大于90%。直接酶受体分析检测结果显示,当纯化受体蛋白1∶500稀释包被时,该重组受体与盐酸克伦特罗、沙丁胺醇及莱克多巴胺的酶标记物结合的OD值分别为0.976、0.836和0.728,亲和活性依次降低。β_2AR蛋白在无细胞体系中的成功表达,为研发基于受体的β激动剂多残留快速检测技术提供了理论支持。
According to the codon preference of Escherichia coli,theβ2 adrenergic receptor（β2AR）gene sequence was designed and synthesized.Then the E.coli-based cell-free protein synthesis（CFPS）system and Magne HisTMNi-Particles were used for efficient expression and purification.The purified receptor was identified by SDS-PAGE analysis and enzyme-linked receptor assays（ELRA）.The results indicated that the codon adaptation index（CAI）of the optimizedβ2AR gene was0.96 and its GC content was decreased from 58%to 46.17%,favoring its expression in E.coli.The optimal Mg2＋concentration in the expression system was 22 mmol/L,leading to the maximum protein expression of 1 250μg/m L.SDS-PAGE revealed the specific band of the purified protein of 47 k Da with a purity of over 90%as expected.The results of direct ELRA showed that when the plates were coated with the receptor at 1：500 dilution,the OD values of the purified receptor binding to three horse radish peroxidase（HRP）-β-agonists decreased in the following order：clenbuterol,salbutamol,and ractopamine,which were 0.976,0.836 and 0.728,respectively.β2AR was successfully synthesized by using the CFPS system,which will provide a theoretical and practical foundation for the rapid multi-residue determination ofβ-agonists based on the receptor.
作者
王健
刘媛
王玮
刘洋
韩正政
曲丽洁
杨立亭
王若敏
WANG Jian;LIU Yuan;WANG Wei;LIU Yang;HAN Zhengzheng;QU Lijie;YANG Liting;WANG Ruomin(Hebei Key Laboratory of Quality ＆ Safety Analysis-Testing for Agro-Products and Food,Food Safety Research Center,Hebei North University,Zhangjiakou 075000,China;Institute of Food Science,College of Agriculture and Forestry Science and Technology,Hebei North University,Zhangjiakou 075000,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2018年第12期179-184,共6页
Food Science
基金
河北省高等学校科学技术研究青年基金项目(QN2018264)
河北北方学院博士科研启动基金项目(201706)
关键词
Β2肾上腺素受体
密码子优化
大肠杆菌无细胞合成体系
表达
β激动剂检测
β2 adrenergic receptor
codon optimization
E. coli-based cell-free protein synthesis system
expression
detection of β-agonists
