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KMID : 0545120190290091470
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Journal of Microbiology and Biotechnology
2019 Volume.29 No. 9 p.1470 ~ p.1477
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Expression of Codon Optimized β2-Adrenergic Receptor in Sf9 Insect Cells for Multianalyte Detection of β-Agonist Residues in Pork
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Liu Yuan
Wang Jian
Liu Yang
Yang Liting
Zhu Xuran
Wang Wei
Zhang Jiaxiao
Wei Dong
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Abstract
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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.
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KEYWORD
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β2-adrenergic receptor, Sf9 cells, codon optimization, β-agonist, receptor-based assay, pork
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