Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression of Codon Optimized β2-Adrenergic Receptor in Sf9 Insect Cells for Multianalyte Detection of β-Agonist Residues in Pork

  • Liu, Yuan (Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food, Hebei North University) ;
  • Wang, Jian (Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food, Hebei North University) ;
  • Liu, Yang (College of Agriculture and Forestry, Hebei North University) ;
  • Yang, Liting (College of Agriculture and Forestry, Hebei North University) ;
  • Zhu, Xuran (Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food, Hebei North University) ;
  • Wang, Wei (College of Agriculture and Forestry, Hebei North University) ;
  • Zhang, Jiaxiao (Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food, Hebei North University) ;
  • Wei, Dong (Hebei Key Laboratory of Quality and Safety Analysis-Testing for Agro-Products and Food, Hebei North University)
  • Received : 2019.06.12
  • Accepted : 2019.08.17
  • Published : 2019.09.28
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Abstract

${\beta}_2$-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 receptorbased assays, indicating that the genetically modified ${\beta}_2-AR$ would have great application potential in detection of ${\beta}$-agonist residues.

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References

  1. Pulce C, Lamaison D, Keck G, Bostvironnois C, Nicolas J, Descotes J. 1991. Collective human food poisonings by clenbuterol residues in veal liver. Vet. Hum. Toxicol 33: 480-481.
  2. Li Y, Lu S, Liu Z, Sun L, Guo J, Hu P, et al. 2015. A monoclonal antibody based enzyme-linked immunosorbent assay for detection of phenylethanolamine A in tissue of swine. Food Chem. 2015; 167: 40-44. https://doi.org/10.1016/j.foodchem.2014.06.085
  3. Hoey AJ, Matthews ML, Badran TW, Pegg GG, Sillence MN. 1995. Cardiovascular effects of clenbuterol are beta 2-adrenoceptor-mediated in steers. J. Anim. Sci. 73: 1754-1765. https://doi.org/10.2527/1995.7361754x
  4. Bolera DD, Shrecka AL, Faulknera DB, Killefera J, McKeitha FK, Hommb JW, et al. 2012. Effect of ractopamine hydrochloride (Optaflexx) dose on live animal performance, carcass characteristics and tenderness in early weaned beef steers. Meat Sci. 92: 458-463. https://doi.org/10.1016/j.meatsci.2012.05.011
  5. The Ministry of Agriculture of China. List of banned animal feed and drinking water substances. Bulletin of the Ministry of Agriculture of the People's Republic of China No. 1519 2010.
  6. European Commission. Council Directive 96/22/EC of 29 April 1996 concerning the prohibition on the use in stockfarming of certain substances having a hormonal orthyrostatic action and of beta-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Official Journal of the European Commission 1996; L125(23): 3-9.
  7. Yao X, Yan P, Tang Q, Deng A, Li J. 2013. Quantum dots based electrochemiluminescent immunosensor bycoupling enzymatic amplification for ultrasensitive detection ofclenbuterol. Anal. Chim. Acta 798: 82-88. https://doi.org/10.1016/j.aca.2013.08.029
  8. Garcia P, Paris AC, Gil J, Popot MA, Bonnaire Y. 2011. Analysis of ${\beta}$-agonists by HPLC/ESI-MS(n) in horse doping control. Biomed. Chromatogr. 25: 147-154. https://doi.org/10.1002/bmc.1562
  9. Du W, Zhao G, Fu Q, Sun M, Zhou H, Chang C. 2014. Combined microextraction by packed sorbent and high-performance liquid chromatography-ultraviolet detection for rapid analysis of ractopamine in porcine muscle and urine samples. Food Chem. 145: 789-795. https://doi.org/10.1016/j.foodchem.2013.08.094
  10. Yang S, Liu X, Xing Y, Zhang D, Wang S, Wang X, et al. 2013. Detection of clenbuterol at trace levels in doping analysis using different gas chromatographic-mass spectrometric techniques. J. Chromatogr. Sci. 51: 436-445. https://doi.org/10.1093/chromsci/bms160
  11. Caban M, Migowska N, Stepnowski P, Kwiatkowski M, Kumirska J. 2012. Matrix effects and recovery calculations in analyses of pharmaceuticals based on the determination of ${\beta}$-blockers and ${\beta}$-agonists in environmental samples. J. Chromatogr. A 1258: 117-127. https://doi.org/10.1016/j.chroma.2012.08.029
  12. Stefano VD, Pitonzo R, Giaccone V, Alongi A, Macaluso A, Cicero N, et al. 2017. Ferrantelli. Analysis of ${\beta}$2-agonists in cattle hair samples using a rapid UHPLC-ESI-MS/MS method. Nat. Prod. Res. 31: 482-486. https://doi.org/10.1080/14786419.2016.1190718
  13. Suo D, Wang R, Wang P, Fan X, Su X. 2017. Pseudo template molecularly imprinted polymer for determination of 14 kind of ${\beta}$-agonists in animal urine by ultra-highperformance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 1526: 23-30. https://doi.org/10.1016/j.chroma.2017.09.076
  14. Nguyen TA, Pham TN, Doan TT, Ta TT, Saiz J, Nguyen TQ, et al. 2014. Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of ${\beta}$-agonists in pharmaceutical and pig-feed samples. J. Chromatogr. A 1360: 305-311. https://doi.org/10.1016/j.chroma.2014.07.074
  15. Gao F, Wu M, Zhang Y, Wang G, Wang Q, He P, et al. 2014. Sensitive determination of four ${\beta}$2-agonists in pig feed by capillary electrophoresis using on-line sample preconcentration with contactless conductivity detection. J. Chromatogr. B 973: 29-32. https://doi.org/10.1016/j.jchromb.2014.10.004
  16. Jiang D, Cao B, Wang M, Yang H, Zhao K, Li J, et al. 2017. Development of a highly sensitive and specific monoclonal antibody based enzyme-linked immunosorbent assay for the detection of a new ${\beta}$-agonist, phenylethanolamine A, in food samples. J. Sci. Food Agric. 97: 1001-1009. https://doi.org/10.1002/jsfa.7826
  17. Han S, Zhou T, Yin B, He P. 2018. Gold nanoparticle-based colorimetric ELISA for quantification of ractopamine. Mikrochim. Acta 185: 210. https://doi.org/10.1007/s00604-018-2736-3
  18. Regiart M, Escudero LA, Aranda P, Martinez NA, Bertolino FA, Raba J. 2015. Copper nanoparticles applied to the preconcentration and electrochemical determination of ${\beta}$-adrenergic agonist: an efficient tool for the control of meat production. Talanta 135: 138-144. https://doi.org/10.1016/j.talanta.2014.12.026
  19. Liu Y, Lu Q, Hu X, Wang H, Li H, Zhang Y, et al. 2017. A Nanosensor based on carbon dots for recovered fluorescence detection clenbuterol in pork samples. J. Fluoresc. 27: 1847-1853. https://doi.org/10.1007/s10895-017-2122-2
  20. Zhang G, Tang Y, Shang J, Wang Z, Yu H, Du W, et al. 2015. Flow-injection chemiluminescence method to detect a ${\beta}$2 adrenergic agonist. Luminescence 30: 102-109. https://doi.org/10.1002/bio.2698
  21. Li M, Yang H, Li S, Zhao K, Li J, Jiang D, et al. 2014. Ultrasensitive and quantitative detection of a new ${\beta}$-agonist phenylethanolamine A by a novel immunochromatographic assay based on surface-enhanced Raman scattering (SERS). J. Agric. Food Chem. 62: 10896-10902. https://doi.org/10.1021/jf503599x
  22. Helbo V, Vandenbroeck M, Maghuin-Rogister G. 1994. Development of a radioreceptor assay for ${\beta}$2 adrenergic agonists. Arch. Lebensmittelhyg 45: 57-61.
  23. Danyi S, Degand G, Duez C, Granier B, Maghuin-Rogister G, Scippo ML. 2007. Solubilisation and binding characteristics of a recombinant beta2-adrenergicreceptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals. Anal. Chim. Acta 589(2): 159-165. https://doi.org/10.1016/j.aca.2007.02.057
  24. Meenagh SA, Elliott CT, Buick RK, Izeboud CA, Witkamp RF. 2001. The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells. Analyst 126: 491-494. https://doi.org/10.1039/b008407g
  25. Boyd S, Heskamp HH, Bovee TF, Nielen MW, Elliott CT. 2009. Development, validation and implementation of a receptor based bioassay capable of detecting a broad range of ${\beta}$-agonist drugs in animal feeding stuffs. Anal. Chim. Acta 637: 24-32. https://doi.org/10.1016/j.aca.2008.09.035
  26. Wang J, She Y, Wang M, Jin M, Li Y, Wang J, et al. 2015. Multiresidue method for analysis of ${\beta}$-agonists in swine urine by enzyme linked receptor assay based on ${\beta}$2-adrenergic receptor expressed in HEK293 cells. PLoS One 10: e0139176. https://doi.org/10.1371/journal.pone.0139176
  27. Wang J, Liu Y, Zhang J, Han Z, Wang W, Liu Y, et al. 2017. Cell-free expression, purification, and characterization of the functional ${\beta}$2-Adrenergic receptor for multianalyte detection of ${\beta}$-Agonists. Biochemistry (Moscow) 82: 1346-1353. https://doi.org/10.1134/S0006297917110128
  28. Cheng G, Li F, Peng D, Huang L, Hao H, Liu Z, et al. 2014. Development of an enzyme- linked-receptor assay based on Syrian hamster ${\beta}$2-adrenergic receptor for detection of ${\beta}$-agonists. Anal. Biochem. 459: 18-23. https://doi.org/10.1016/j.ab.2014.05.005
  29. Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, et al. 2007. Crystal structure of the human ${\beta}$2-adrenergic G-protein-coupled Receptor. Nature 450: 383-387. https://doi.org/10.1038/nature06325
  30. Parker E M, Kameyama K, Higashijima T, Ross EM. 1991. Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells. J. Biol. Chem. 266: 519-527. https://doi.org/10.1016/S0021-9258(18)52467-4