›› 2018, Vol. 45 ›› Issue (7): 1768-1774.doi: 10.16431/j.cnki.1671-7236.2018.07.005

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Construction of the Eukaryotic Expression Plasmid for Mutants of Porcine Orexin 2 Receptor and Their Effect on cAMP Signaling Pathways

LIU Min1,2, MIN Tianqi1,2, ZHANG Haijie1,2, XIE Huajie1,2, CUI Zihe1,2, WANG Zhiqiang1,2   

  1. 1. Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
    2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
  • Received:2018-01-25 Online:2018-07-20 Published:2018-07-20

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Abstract:

This study were aimed to construct the eukaryotic expression vectors for porcine orexin 2 receptor (pOX2R) mutants and explore the difference in constitutive activity between wild type and mutants.We used the eukaryotic expression vector pcDNA3.1(+)/pOX2R-myc WT as the template to construct four mutants,named pcDNA3.1(+)-myc/pOX2R-P10S,pcDNA3.1(+)-myc/pOX2R-P11T,pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I,and then transient transfected the plasmids into HEK293T cell.The basal activities of pOX2R wild-type and mutants were determined using dual luciferase reporter assays,the intracellular cAMP levels were measured at different concentrations of agonists,the wild-type and mutants were stimulated with the endogenous agonists orexin A (OXA) and orexin B (OXB),respectively.The results showed that the recombinant plasmid pcDNA3.1(+)/pOX2R-myc-P10S,pcDNA3.1(+)-myc/pOX2R-P11T,pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I were constructed successfully.The 10,11,308 and 401 amino acids of the pOX2R were successfully mutated to serine,threonine,isoleucine and valine,respectively.There were no significant difference between reconstruction plasmid of pcDNA3.1(+)-myc/pOX2R WT and four mutants in basal activity (P>0.05).It showed that there was no significant effect of amino acid mutations at 4 sites on the basal expression signal of their receptors.Compared with the wild-type receptor,there was no significant difference in the response of the mutant receptor to OXB (P>0.05),but the EC50 of the mutant P10S,P11T and T401I to agonist OXA was significantly lower (P<0.05),there was no significant difference in Rmax (P>0.05).The mutants (P10S,P11T and T401I) might be affect the binding between agonist OXA ligand and receptor,and reduce the agonistic effect of the agonist.The study provided foundation for the further exploration of the function of pOX2R.The results laid the foundation for further study of pOX2R function in vitro.

Key words: porcine; OX2R gene; mutant; cAMP

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