β_2肾上腺素受体基因的改造及在无细胞合成体系中的表达  

Gene Modification and Expression of β_2 Adrenergic Receptor in a Cell-Free Protein Synthesis System

作　　者：王健 刘媛[1,2] 王玮 刘洋[1] 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)

机构地区：[1]河北北方学院食品安全研究中心河北省农产品食品质量安全分析检测重点实验室,河北张家口075000 [2]河北北方学院农林科技学院食品研究所,河北张家口075000

出　　处：《食品科学》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)

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.

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 

分 类 号：TS201.6[轻工技术与工程—食品科学]