通过密码子优化、昆虫杆状病毒表达系统(Bac-to-Bac)构建、表达条件筛选及镍柱亲和层析,研究β2肾上腺素受体(β2 adrenergic receptor,β2AR)基因(β2AR)在昆虫细胞Sf9中的高效表达及纯化策略.方法:人工合成改造后的β2AR基因,将其克隆至转移载体pFastBac1中,构建重组杆状病毒表达质粒pFastBac l-β2AR',转染昆虫细胞Sf9,优化表达条件,采用镍离子亲和层析法纯化重组蛋白并进行活性鉴定.结果:适宜的表达条件为感染细胞使用的感染复数5、感染后表达时间48 h,Western blot分析显示在47 kD左右处出现清晰的特异性条带,与预期结果一致.纯化的受体蛋白纯度大于90％,活性鉴定结果显示该受体蛋白可特异性吸附盐酸克伦特罗、沙丁胺醇及莱克多巴胺3种β激动剂的酶标记物,OD值分别为0.983、0.947和0.912.结论:本研究实现了β2AR受体在Sf9细胞中的表达,且纯化后的受体蛋白保持了较好的β激动剂亲和活性,为利用β2AR受体开展β激动剂多残留快速检测技术提供了依据.%Codon optimization,construction of recombinant baculovirus system (Bac-to-Bac),screening of optimal expression conditions and Ni-chelating affinity chromatography were used to develop an efficient strategy for the expression and purification of β2 adrenergic receptor (β2AR) gene in Sf9 cells.The modified β2AR gene was artificially synthesized and cloned to the transfer vector pFastBac 1 to construct the recombinant baculovirus expression plasmid pFastBacl-β2AR'.Then Sf9 cells were transfected with the recombinant plasmid.The expression conditions were optimized.The expression product was purified by Ni-NTA-affinity chromatography and its ligand binding affinity was determined by enzyme-linked immunosorbent assay (ELISA).The results showed that the optimal expression conditions were as follows:multiplicity of infection (MOI) of the infected cells,5;and expression time,48 h.In the Western blot analysis,a single band with an apparent molecular mass of 47 kD appeared as expected.After purification,the recombinant protein was more than 90％ pure.The ligand binding assay indicated that it could specifically bind to all three horseradish peroxidase (HRP)-β-agonists:clenbuterol,salbutamol,ractopamine,and the OD values obtained from ELISA were 0.983,0.947 and 0.912,respectively.In this paper,the efficient expression ofβ2AR in Sf9 cells was accomplished.Furthermore,the purified receptor protein remained better binding affinity to β-agonists,laying a foundation for developing a rapid multi-residue assay for the determination ofβ-agonists with β2AR.
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