食品科学2017，Vol.38Issue(20)：34-39,6.DOI:10.7506/spkx1002-6630-201720006
β2肾上腺素受体基因在Sf9细胞中的表达及纯化
Expression and Purification of β2 Adrenergic Receptor in Sf9 Cells
摘要
Abstract
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.关键词
β2肾上腺素受体/杆状病毒表达载体/Sf9细胞/表达/纯化Key words
β2 adrenergic receptor/baculovirus expression vector/Sf9 cells/expression/purification分类
轻工纺织引用本文复制引用
王健,刘媛,张俊花,韩正政,兰凤英..β2肾上腺素受体基因在Sf9细胞中的表达及纯化[J].食品科学,2017,38(20):34-39,6.基金项目
河北北方学院博士启动基金项目(201706) （201706）
公益性行业(农业)科研专项(201203094) （农业）
