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Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

Wang J, She Y, Wang M, Jin M, Li Y, Wang J, Liu Y - PLoS ONE (2015)

Bottom Line: After activity identification, the recombinant receptor was used in the development of direct competitive ELRA.The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively.ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

View Article: PubMed Central - PubMed

Affiliation: Institute of Quality Standards and Testing Technology for Agro-products of CAAS, Key Laboratory of Agro-Product Quality and Safety, Ministry of Agriculture, Beijing, 100081, P. R. China; Department of Food Science, Hebei North University, Zhangjiakou, 075000, P. R. China.

ABSTRACT
A novel enzyme-linked receptor assay (ELRA) based on β2-adrenergic receptor (β2-AR) has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists) in urine. Human embryonic kidney cells (HEK293) were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

No MeSH data available.


Related in: MedlinePlus

Agarose gel electrophoresis analysis of 3 combinant expression plasmid.The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β2-AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.
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pone.0139176.g001: Agarose gel electrophoresis analysis of 3 combinant expression plasmid.The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β2-AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.

Mentions: Recombinant plasmid of pTriEx-1.1 Hygro-β2-AR was constructed successfully after verification of PCR (Fig 1, lanes 3-4), double enzyme digestion (Fig 1, lanes 1-2), and sequencing. As the plamid could be used to test the expression in 3 systems of E. coli, insect, and mammalian cells, the expression study was performed first in E. coli BL21 (DE3); however, the expression level indicated by SDS-PAGE and ELISA was very low (data not shown). Consequently, transient expression of β2-AR gene in HEK293 cells was explored and the approximate densities ranged from 17 to 23 pmol/mg of membrane protein. This expression level was still not completely satisfactory, but could basically meet the requirement of its exploration in the development of a new detection method. In order to obtain higher amount of the recombinant receptor, site-specific mutagenesis of β2-AR gene or optimization of expression vector would be useful for futher reseach. Western blot was probed with anti-His tag monoclonal antibody, and the results revealed that β2-AR expressed in HEK293 cells appeared in electrophoresis as diffuse bands with an apparent molecular mass in the range of 47-55 kDa (Fig 2, lane 1), compared with the cellular proteins of nontransfected HEK293 cells (Fig 2, lane 2). The molecular mass range of the target protein was close to the molecular mass (46.73 kDa) calculated from the amino acid sequence of monomeric β2-AR. One of the interpretation of the result was that glycosylation contributed to 4 and 8 kDa mass increment [27]. To reach the highest expression level of recombinant β2-AR, the optimal expression time was determined as 72 hours after transfection (data not shown).


Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

Wang J, She Y, Wang M, Jin M, Li Y, Wang J, Liu Y - PLoS ONE (2015)

Agarose gel electrophoresis analysis of 3 combinant expression plasmid.The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β2-AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589316&req=5

pone.0139176.g001: Agarose gel electrophoresis analysis of 3 combinant expression plasmid.The plasmid was confirmed by PCR and double digestion using NcoI and XhoI. Lane 1 was the fragment of recombinant plasmid DNA pTriEx-1.1 Hygro-β2-AR. Lane 2 was the electrophoresis results of digested products containing 2 fragments (6951 bp and 1257 bp). A 3300 bp fragment (lane 3 and lane 4) was amplified by PCR from the recombinant plasmid, which was identical with the sum of the size of target gene and vector sequences between NcoI and XhoI.
Mentions: Recombinant plasmid of pTriEx-1.1 Hygro-β2-AR was constructed successfully after verification of PCR (Fig 1, lanes 3-4), double enzyme digestion (Fig 1, lanes 1-2), and sequencing. As the plamid could be used to test the expression in 3 systems of E. coli, insect, and mammalian cells, the expression study was performed first in E. coli BL21 (DE3); however, the expression level indicated by SDS-PAGE and ELISA was very low (data not shown). Consequently, transient expression of β2-AR gene in HEK293 cells was explored and the approximate densities ranged from 17 to 23 pmol/mg of membrane protein. This expression level was still not completely satisfactory, but could basically meet the requirement of its exploration in the development of a new detection method. In order to obtain higher amount of the recombinant receptor, site-specific mutagenesis of β2-AR gene or optimization of expression vector would be useful for futher reseach. Western blot was probed with anti-His tag monoclonal antibody, and the results revealed that β2-AR expressed in HEK293 cells appeared in electrophoresis as diffuse bands with an apparent molecular mass in the range of 47-55 kDa (Fig 2, lane 1), compared with the cellular proteins of nontransfected HEK293 cells (Fig 2, lane 2). The molecular mass range of the target protein was close to the molecular mass (46.73 kDa) calculated from the amino acid sequence of monomeric β2-AR. One of the interpretation of the result was that glycosylation contributed to 4 and 8 kDa mass increment [27]. To reach the highest expression level of recombinant β2-AR, the optimal expression time was determined as 72 hours after transfection (data not shown).

Bottom Line: After activity identification, the recombinant receptor was used in the development of direct competitive ELRA.The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively.ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

View Article: PubMed Central - PubMed

Affiliation: Institute of Quality Standards and Testing Technology for Agro-products of CAAS, Key Laboratory of Agro-Product Quality and Safety, Ministry of Agriculture, Beijing, 100081, P. R. China; Department of Food Science, Hebei North University, Zhangjiakou, 075000, P. R. China.

ABSTRACT
A novel enzyme-linked receptor assay (ELRA) based on β2-adrenergic receptor (β2-AR) has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists) in urine. Human embryonic kidney cells (HEK293) were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

No MeSH data available.


Related in: MedlinePlus