| ?2 adrenergic receptor(?2AR)belongs to the G protein-coupled receptor family and exists in the smooth muscle of animal body widely.It is related to renin secretion and glycogenolysis.In view of the relevant biological effects of ?2AR,?2-agonists was used as a feed additive for food animals in order to increase lean meat percentage,reduce fat deposition and improve feed conversion rate.Therefore,it also brings about the problem of residual?2-agonists.As a pig breeding country,the detection of ?-receptor agonist residue is very important to public health and safety in our country.There are two mainly existing detection methods which are chromatography and immunological methods,but they can not cope with the emerging new type of P-agonists.In addition,the specificity of the target makes them have some limitations.Receptor assay residue detection method is proposed in recent years to deal with a certain type of residual drug residues.It has been successfully applied to veterinary drugs such as P-lactam residue detection.It has several advantages,such as has a low limit of detection,high sensitivity,short detection time and so on.However,the research on the method of receptor assay residue detection for ?2-agonists is still in the initial stage of exploration.Therefore,this study aims to find a suitable receptor model for receptor assay,lay the foundation of receptor analysis of residual detection methods to ?2-agonists.In this study,the porcine ?2 adrenergic receptor(?2AR)gene and its mutant eukaryotic expression vector were constructed and their expression in HEK293T cells was identified.In this study,pig ?2AR gene was cloned from porcine genomic DNA and subcloned into eukaryotic expression vector pcDNA3.1(+)using homologous recombination technology to construct eukaryotic expression vector pcDNA3.1(+)-?2AR.After adding myc-tag,then named it as myc-pcDNA3.1(+)-?2AR.The ?2AR-D130N,?2AR-C285S and ?2AR-D130N/C285S were constructed by using the eukaryotic expression vector as a template.The constructed eukaryotic expression vectors and mutants were transfected into HEK293T cells,and their expression was verified by Western blot.The results showed that the porcine ?2AR gene was correctly recombined into the pcDNA3.1(+)vector.The results of sequencing showed that the 130th amino acid has been mutate from aspartic acid to asparagine and the 285th amino acid has been mutate from cysteine to serine.Also,we confirmed that the plasmids can express in HEK293T successfully.After verifying that the constructed expression vectors and mutants can be expressed successfully,we researched some of their pharmacological activities.In the present study,the expression vector and the mutant constructed in the previous step were first transfected into HEK293T cells and then incubated with different ?2-agonists(Isoprenaline,Clenbuterol and Salbutamol)and ?2-receptor antagonists(Propranolol)in three different concentrations(10'6M,10-8M and 10-10M)respectively.After 24h,the cells were lysed,the protein was extracted and Western Bolt was used to detect the related pathway proteins.In this study,we successfully constructed the eukaryotic expression vector of porcine ?2AR wild-type and mutant,and verified the expression in HEK293T cells.In the study of pharmacological properties,by studying the proteins' changes of MAPK and PKA signaling pathway with different ?2AR agonists and antagonists,it was found that mutant D130N is in a relatively activated state,The effect of agonist stimulation is more sensitive,so the mutant may be more suitable for use in beta-receptor agonist receptor assay residue assays than the other three ?2ARs involved in this study. |
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