Establishment Of MGluR₂ Stable Cell Line And Its Function Detection

Metabotropic glutamate receptors?mGluRs?is an important neurotransmitter receptor,which mainly exists in mammalian nerve cells,the receptor is a C family members of G protein coupled receptor?G protein coupled receptor GPCRs?.the receptor is divided into three categories according to the receptor protein sequence homology,coupled second messenger,and agonist specificity:type I mGluRs include mGluR₁ and mGluR₅;type II mGluRs include mGluR₂ and mGluR₃;type III mGluRs include mGluR₄,mGluR_6-8.Typically,the receptor is involved in a variety of physiological and pathological activities through the receptor interacting with the natural ligand glutamate.The interaction causes changes in the receptor conformation,resulting in the receptor is active,and then activates the G protein,triggering downstream signals.The results of receptor activation are mainly to regulate synaptic transmission,regulate neuronal excitability.The diversity of receptor subtypes plays a physiological function.which triggers a variety of physiological processes such as:learning,memory,cognitive and other closely related,while the receptor is also widely involved in a variety of neurodegenerative diseases,Which also makes the research workers on the receptor research enthusiasm continued.The function of metabolic glutamate receptors are implementated by very fine regulation,which results in any minor changes,over-activation or excessive inhibition,will lead to a variety of neurological and psychiatric disorders.In addition,the receptor also with the occurrence of cancer,tumor malignant proliferation is also closely related to the cancer involved in a variety of signaling pathways.Studies have found that inhibition of intracellular L-glutamic acid release,through competitive or non-competitive antagonists to inhibit receptor activation can reduce cell proliferation,migration,invasion ability,induced tumor cells such as breast cancer,melanoma,Glioma and other cell apoptosis.In summary,metabolic glutamate receptors and L-glutamic acid-initiated signaling pathways have become very promising drug targets for the treatment of neurodegenerative diseases and tumors.At present,mGluR₂ drug target for drug research and development work has made great progress,the research and development of a variety of drugs have been used for clinical,but the shortcomings is that mGluR₂ as a target to develop some of the clinical application of drugs The process of drug dependence,specificity and other side effects.Therefore,the construction of mGluR₂ as a target to stabilize the cell model,its use in drug screening and research and development,is conducive to scientific research workers on mGluR₂ research,and can improve the mGluR₂ as the target drug screening and research and development progress.In this study,mGluR₂ receptor was selected as the drug target to construct a drug screening model based on the target cell line.The plasmid pOG44,pcDNA5/FRT-Flag-CLIP-mGluR₂-GB₂-C-KKTN and pEGFP-N1-HA-SNAP-mGluR₂-GB₂-CKTN constructed in our laboratory were co-transfected into Flp-in CHO cell lines,Using the corresponding antibiotics to screen out possible monoclonal,and then Ca²⁺signal detection method,obtained mGluR₂ receptor stable expression of cell lines.Then,the metabolizing glutamate receptor and the agonist LY379268 were used to detect the cell line and the stability test.The stable expression vector of mGluR₂ was obtained.In this study,pOG44 is a plasmid that can assist in the integration of exogenous plasmids into the host cell genome and help the foreign gene to stably express with the host cell,whereas the SNAP and CLIP tags can confer a new,characteristic drug to the cell model Screening means,time-resolved fluorescence resonance energy transfer technique?TR-TRFT?.In the process of cell line construction,we constructed pOG44,plasmid pEGFP-N1-HA-SNAP-mGluR₂-GB₁-C-KKTN and CLIP tagged plasmid pcDNA5/FRT-Flag-mgluR₂-GB₂-C-KKTN was transferred into Flp-in CHO cells.G418 and HygromycinB were used to screen,and enzyme-linked immunosorbent assay?ELISA?and second messenger(Ca²⁺)signal detection.Downstream Ca²⁺signal cloned cell lines,and finally built based on the target of mGluR₂ receptor high-throughput drug screening cell line model.In this paper,a high-flux stabilized drug screening model targeting mGluR₂ receptors was established.The advantage of this cell line model is that it can screen small-molecule active compounds,which can be used with higher specificity,better stability,no radioactivity new technology means,greatly improving the development of neurodegenerative diseases drug efficiency.