Journal
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
Volume 29, Issue 9, Pages 1470-1477Publisher
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
DOI: 10.4014/jmb.1906.06022
Keywords
beta(2)-adrenergic receptor; Sf9 cells; codon optimization; beta-agonist; receptor-based assay; pork
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Funding
- Youth Research Foundation of the Education Bureau of Hebei Province, China [QN2018264]
- Doctoral Scientific Research Foundation of Hebei North University [201706]
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beta(2)-adrenergic receptor (beta(2)-AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of beta-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 mu g/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 mu g/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 receptor-based assays, indicating that the genetically modified beta(2)-AR would have great application potential in detection of beta-agonist residues.
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