猪β_2肾上腺素受体的表达、纯化及鉴定

Expression, Purification, and Identification of β_2 Adrenergic Receptors in Pigs

研究了β2肾上腺素受体(β2 adrenergic receptor,β2AR)基因重组表达质粒在人胚肾细胞293(Human embryonic kidney cells293,HEK293)中的瞬时转染及表达产物的纯化和鉴定。将已构建的重组表达质粒瞬时转染HEK293细胞,表达产物制备粗膜蛋白后,采用镍离子亲和层析法纯化得到受体蛋白并进行鉴定。结果表明:重组表达质粒p Tri Ex-1.1 Hygro-β2AR1-418转染HEK293细胞后表达量和蛋白活性较优,最佳表达时间为转染后72 h。SDS-PAGE和Western blot结果显示,在47 ku左右出现了特异性条带,咪唑的最佳洗脱浓度为250 mmol/L,受体蛋白纯度大于80%。活性鉴定表明:受体蛋白与HRP酶标记的3种β激动剂均能特异性结合,检测HRP-克伦特罗、HRP-莱克多巴胺和HRP-沙丁胺醇的OD值分别为0.97、0.91和0.94。通过纯化得到的受体蛋白,约占粗膜蛋白的4.4%。本试验成功获得一定数量纯度和活性较好的β2AR受体蛋白,为受体技术在β激动剂多残留检测中的研发和应用奠定了基础。

A recombinant porcine β_2 adrenergic receptor（β_2AR） expression plasmid was transiently transfected in human embryonic kidney cells 293（HEK293）. The expressed products were purified by crude membrane preparation and Ni-NTA-affinity chromatography to yield the receptor protein, followed by identification. The results showed that the HEK293 cells transfected with the recombinant expression plasmid p Tri Ex-1.1 Hygro-β_2AR1-418 exhibited high expression level and protein activity, and the optimal expression time was 72 h after transfection. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and western blotting indicated a specific band occurred at 47 k Da, the optimal imidazole concentration for elution was 250 mmol/L, and the purity of the receptor protein was more than 80%. The activity assay revealed that the recombinant receptor could specifically bind all three HRP-β-agonists, and the optical density（OD） values obtained for the detection of HRP-CBL, HRP-RAC, and HRP-SAL were 0.97, 0.91, and 0.94, respectively. The purified receptor protein accounted for 4.42% of crude membrane protein. In brief, β_2AR receptor protein with ideal purity and activity was obtained, laying a foundation for the development and application of this receptor technique in the multi-residue determination of β-agonists.