摘要：
依据大肠杆菌密码子偏好性,设计合成β2肾上腺素受体(β2adrenergic receptor,β2AR)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MagneHis? 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β2AR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Mg2+浓度为22?mmol/L,此时表达量为1 250 μg/mL.SDS-PAGE分析显示,纯化蛋白在47?kDa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.β2AR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, the β 2adrenergic receptor (β 2AR) gene sequence was designed and synthesized. Then the E. coli-based cell-free protein synthesis (CFPS) system and MagneHis?Ni-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 optimized β2AR gene was 0.96 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 1 250 μg/mL. SDS-PAGE revealed the specific band of the purified protein of 47 kDa 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 of β-agonists based on the receptor.
