Gene Cloning Of Pig Orexin-2Receptor And Its Ligands Affect CAMP Signaling Pathways

Orexin is a neuropeptide highly expressed in many different regions of the brain. These peptides exert their actions via two different G protein-coupled receptors, namely OX1and OX2Orexin receptors. Orexin is an important signaling molecule can regulate energy dynamic balance. They have been implicated in feeding, energy homeostasis, neuroendocrine function, cardiovascular control, pain, and sleep—ake cycles. OX1R and OX2R are coupled to Gq/11proteins that activate the PLC/IP3pathway, leading to a transient increase of intracellular calcium. The OX2R can also be coupled to Gs and Gi pathways, leading to increase or decrease of intracellular cAMP.In order to study the effects of OX2R on the regulation of pig’growth and energy, this study was conducted for the molecular cloning and construction of eukaryotic expression vector of OX2R gene. And then HEK293T cells were transiently transfected with the plasmids of OX2R and with three CRE response element luciferase genes together by lipidosome method. The luciferase was measured by GloMax-Multi after different of ligands act on cells. The results were as follows:1. The1332bp pOX2R gene was obtained by RT-PCR from the hypothalamus of pig and it was cloned into the vector pcDNA3.1(+). The myc-tag sequence recombinant plasmid pcDNA3.1(+)-myc/pOX2R was successfully constructed.2. HEK293T cells were transiently transfected with the plasmids of OX2R gene by lipofectamine method. The pOX2R fused protein with myc-tag could be detected in HEK293T cells by Western blot.3. With the ratio of1:5, the plasmid pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro were transfected HEK293T cells by lipofectamine method.10-4M、10-7M and10-10M three different concentrations of TCS OX229(the antagonist of OX2R) with a series concentrations of Orexin A or Orexin B act on the cells respectively where the OX2R and luciferase gene were expressed. The results showed that, in a dose effect relationship of the concentration range, the cAMP level has a good trend.