2019 ; Vol.29-9: 1470~1477
|Author||Yuan Liu, Jian Wang, Yang Liu, Liting Yang, Xuran Zhu, Wei Wang, Jiaxiao Zhang, Dong Wei|
|Place of duty||Hebei Key Laboratory of Quality & Safety Analysis-Testing for Agro-Products and Food , Hebei North University, P.R. China,College of Agriculture and Forestry, Hebei North University, P.R. China,Zhangjiakou Key Laboratory of Quality & Safety for Charactenistics Agro-Products,Hebei North University, P.R. China|
|Title||Expression of Codon Optimized β2-Adrenergic Receptor in Sf9 Insect Cells for Multianalyte Detection of β-Agonist Residues in Pork|
J. Microbiol. Biotechnol.2019 ;
|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|
|Key_word||β2-adrenergic receptor, Sf9 cells, codon optimization, β-agonist, receptor-based assay, pork|
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.
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.
Hoey AJ, Matthews ML, Badran TW, Pegg GG, Sillence MN. 1995. Cardiovascular effects of clenbuterol are beta 2adrenoceptor-mediated in steers. J. Anim. Sci. 73: 1754-1765.
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.
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. 15192010.
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.
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.
Garcia P, Paris AC, Gil J, Popot MA, Bonnaire Y. 2011. Analysis of β-agonists by HPLC/ESI-MS(n) in horse doping control. Biomed. Chromatogr. 25: 147-154.
Du W, Zhao G, Fu Q, Sun M, Zhou H, Chang C. 2014. Combined microextraction by packed sorbent and highperformance liquid chromatography-ultraviolet detection for rapid analysis of ractopamine in porcine muscle and urine samples. Food Chem. 145: 789-795.
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.
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 β-blockers and β-agonists in environmental samples. J. Chromatogr. A 1258: 117-127.
Stefano VD, Pitonzo R, Giaccone V, Alongi A, Macaluso A, Cicero N, et al. 2017. Ferrantelli. Analysis of β2-agonists in cattle hair samples using a rapid UHPLC-ESI-MS/MS method. Nat. Prod. Res. 31: 482-486.
Suo D, Wang R, Wang P, Fan X, Su X. 2017. Pseudo template molecularly imprinted polymer for determination of 14 kind of β-agonists in animal urine by ultra-highperformance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 1526: 23-30.
Nguyen TA, Pham TN, Doan TT, Ta TT, Sáiz J, Nguyen TQ, et al. 2014. Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of β-agonists in pharmaceutical and pig-feed samples. J. Chromatogr. A 1360: 305-311.
Gao F, Wu M, Zhang Y, Wang G, Wang Q, He P, et al. 2014. Sensitive determination of four β2-agonists in pig feed by capillary electrophoresis using on-line sample preconcentration with contactless conductivity detection. J. Chromatogr. B 973:29-32.
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 β-agonist, phenylethanolamine A, in food samples. J. Sci. Food Agric. 97: 1001-1009.
Han S, Zhou T, Yin B, He P. 2018. Gold nanoparticle-based colorimetric ELISA for quantification of ractopamine. Mikrochim. Acta 185: 210.
Regiart M, Escudero LA, Aranda P, Martinez NA, Bertolino FA, Raba J. 2015. Copper nanoparticles applied to the preconcentration and electrochemical determination of βadrenergic agonist: an efficient tool for the control of meat production. Talanta 135: 138-144.
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.
Zhang G , T ang Y , S hang J , W ang Z , Y u H , Du W , et al. 2015. Flow-injection chemiluminescence method to detect a β2 adrenergic agonist. Luminescence 30: 102-109.
Li M, Yang H, Li S, Zhao K, Li J, Jiang D, et al. 2014. Ultrasensitive and quantitative detection of a new β-agonist phenylethanolamine A by a novel immunochromatographic assay based on surface-enhanced Raman scattering (SERS). J. Agric. Food Chem. 62: 10896-10902.
Helbo V, Vandenbroeck M, Maghuin-Rogister G. 1994. Development of a radioreceptor assay for β2 adrenergic agonists. Arch. Lebensmittelhyg 45: 57-61.
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.
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.
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 β-agonist drugs in animal feeding stuffs. Anal. Chim. Acta 637: 24-32.
Wang J, She Y, Wang M, Jin M, Li Y, Wang J, et al. 2015. Multiresidue method for analysis of β-agonists in swine urine by enzyme linked receptor assay based on β2adrenergic receptor expressed in HEK293 cells. PLoS One 10:e0139176.
Wang J, Liu Y, Zhang J, Han Z, Wang W, Liu Y, et al. 2017. Cell-free expression, purification, and characterization of the functional β2-Adrenergic receptor for multianalyte detection of β-Agonists. Biochemistry (Moscow) 82: 1346-1353.
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 β2-adrenergic receptor for detection of β-agonists. Anal. Biochem. 459: 18-23.
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, et al. 2007. Crystal structure of the human β2-adrenergic G-protein-coupled Receptor. Nature 450: 383-387.
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.