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Expression of Codon Optimized β2-Adrenergic Receptor in Sf9 Insect Cells for Multianalyte Detection of β-Agonist Residues in Pork

Journal of Microbiology and Biotechnology 2019년 29권 9호 p.1470 ~ 1477
 ( Liu Yuan ) - Hebei North University College of Agriculture and Forestry

 ( Wang Jian ) - Hebei North University College of Agriculture and Forestry
 ( Liu Yang ) - Hebei North University College of Agriculture and Forestry
 ( Yang Liting ) - Hebei North University College of Agriculture and Forestry
 ( Zhu Xuran ) - Hebei North University Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food
 ( Wang Wei ) - Hebei North University College of Agriculture and Forestry
 ( Zhang Jiaxiao ) - Hebei North University Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food
 ( Wei Dong ) - Hebei North University Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food

Abstract


β2-adrenergic receptor (β2-AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of β-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and 59.57 μg/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 μg/l and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified β2-AR would have great application potential in detection of β-agonist residues.

키워드

β2-adrenergic receptor; Sf9 cells; codon optimization; β-agonist; receptor-based assay; pork
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