Direct Competitive Enzyme-linked Recombinant Receptor Analysis For Detection Of β Adrenergic Agonists

At present, the illegal use of β-adrenergic agonists(β-agonists for short) is still one of the main factors which influence animal food safety in our country. Especially the “low dose” cocktail and continuous turn over of β-agonists have brought great challenges to supervision. Thus, it is urgently needed to establish a rapid screening approach for multi-residues determination of β-agonists. In this study based on the, porcine β2AR gene was cloned and the optimal expression system was choosed. The purified receptor protein was used as a recognition element in order to establish a direct competitive enzyme-linked receptor assay for detection of Clenbuterol(CBL), Ractopamine(RAC),Salbutamol(SAL) and so on. The main contents and results are summarized as follows:1. Gene cloning of the pig β2AR and sequence analysis. By sequencing, the two fragments were both 1257 bp, encoding 418 amino acids. Both of them had extremely high identities of nucleotide and amino acid to that of the published pig β2AR. Furthermore, all of the amino acids at the ligand binding sites were cloned correctly. Bioinformation analysis showed that the deduced protein was accord with the structural characteristics of β2AR. Homology comparison combined with phylogenetic tree analysis indicated that there were high homology of β2AR between pig and other species, and Sus scrofa is the most close to Pecan tajacu2. Screening of the optimal expression system and condition. The wild-type and modified β2AR gene were expressed in Pichia pastoris X-33, Escherichia Coli BL21 and mammalian cells HEK293, respectively. Then the expression effect was compared among them and the expression condition was optimized. After the expression level was detected by SDS-PAGE and Western blot, and the activity of the protein was identified by ELISA, the results showed that the HEK293 cells transfected with p Tri Ex-1.1 Hygro-β2AR1-418 exhibited higher expression level, with the highest affinity of the crude protein from it. The OD values obtained from the ELISA for detection 3 HRP-β-agonists were 0.872, 0.652 and 0.848. Therfore, it was choosed as the optimal expression system and the optimal expression time was 72 h after transfection.3. Preparetion and purification of the receptor protein. By lysis and membrane dissolution, the crude membrane protein was prepared, followed by purification of the receptor protein using Ni-NTA affinity chromatography. The results of SDS-PAGE and Western blot indicated that the optimal imidazole concentration was 250 mmol/L, a specific bank was seen at 47 KD, and the purity of the protein was more than 80%. The activitiy assay revealed that the recombinant receptor could bind all 3 HRP-β-agonists, and ranking based on their binding affinity was HRP-CBL>HRP-SAL>HRP-RAC. The yield of the receptor protein was 160 μg/4 pates(15 cm), 4.4% of crude membrane.4. The ELRA method for detection of β-agonists was established based on the purified receptor protein. The optimal working conditions were as flows: the receptor protein and HRP-CBL were respectively diluted in the ratio of 1: 10 and 1: 500. Coating buffer and blocking buffer were respectively carbonate-buffered solution(0.05 mol/L, p H 9.6) and 1% BSA, with the proper blocking condition was 4℃ overnight. Both of ELRA based on receptor protein and crude protein could realize the quantitative detection of the 3 β-agonists. Moreover, the former obviously presented better linear relation in the concentration range of 1 μg/L~1000 μg/L, with R2 were 0.9933, 0.9953 and 0.9966, respectively. Methodological evaluation showed that LOD of the method was 4.49 μg/L; the IC50 values of CBL, SAL, and RAC calculated from the linear regression equations were respectively 32.13, 71.43, and 96.11 μg/L, and RAC and SAL exhibited the cross reactivity with CBL was 33.4% and 45.0%, respectively; the average recovery rates of CBL, RAC, and SAL were 68.2%, 60.3%, and 65.5%, respectively; all CVs were within 15%, indicating an ideal variation extent.In conclusion, ELRA for detection of 3 β-agonists in urine was established, providing scientific basis for the development and application of β2AR in multi-residues determination of β-agonists.