Fusion Gene Of GPCRs-Gila And Construction Of CHO-pcDNA3.1(+)-GPR91Engineeirng Cells

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors,which function through G protein mediation and are involved in a wide range ofpathophysiological processes. GPCRs can mediate cell communications and signaltransduction, play important physiological roles and pathological significances. As aresult, GPCRs have been the most important targets for drug research and development.To date, the number of drugs targeting GPCRs constitutes about40%-50%of thecommercially available drugs.In a total of about900GPCRs, about100members have not been identified in thatendogenous ligands and function which are called orphan G protein-coupled receptors(oGPCRs). Due to the important roles of GPCRs in the pathophysiology and drugresearch, identification of oGPCR endogenous ligands and its mechanism of action hasbecome an important subject for the study of GPCRs. The identification of the specificligands for oGPCRs has been called “deorphaning” Once oGPCRs deorphansuccessfully, which have great significance for the elucidation of the related diseasepathogenesis and drug research found.In order to efficiently identify GPCRs specific ligands and their mechanisms of action,we have designed the present study. It mainly consists of two parts: the first part focuseson the gene fusion protein of three GPCRs members with the G protein α subunitseperately, which can be used to screen the ligands of GPCRs; the second part is toconstruct CHO-pcDNA3.1(+)-GPR91engineering cells, by which the mechanisms of action of GPR91will be explored in-depth.In the first part of this experiment, GPR91, GPR119and GPR84were fused with Gi1αseperately. Firstly, the full-length of open reading frames of the above four moleculeswere obtained by molecular cloning, and they were subcloned to the pMD18-T vector toconstruct the recombinant plasmid. Using the recombinant plasmid as template,corresponding the GPCRs and Gi1α fusion genes were amplified by overlap extensionPCR. Then, the fusion genes ware cloned into the donor plasmid pFASTBac1to obtainthe recombinant pFASTBac1-GPCRs-Gi1α. By transfection into the E.coli DH10Baccompetent using pFASTBac1-GPCRs-Gi1α, the recombinant pBacmid-oGPCR-Gi1αwas successfully constructed with Bac-to-Bac baculovirus expression system indicatedby specific transposition.In the second part of this experiment, we cloned the open reading frames of GPR91intovector pcDNA3.1(+), and then transferred the recombinant vector into CHO cells,obtained the positive clones through screening, and successfully constructed theCHO-pcDNA3.1(+)-GPR91engineering cells. RT-PCR and Western-blot analysisshowed that the CHO-pcDNA3.1(+)-GPR91engineering cells we could express theGPR91mRNA and protein. Stimulated with succinate, an endogenous ligand of GPR91,the engineering cells can be detected the intracellular Ca2+concentration alterationsignificantly by using the fluorescent probes and confocal imaging system. It isindicated that the CHO-pcDNA3.1(+)-GPR91engineering cells can be receptor agoniststimulated, and can be used for the GPR91agonist or antagonist screening.