Establishment Of Screening Model For Cannabiniod Receptor Two Agonists And Screening Of Potential Activity Drug

Background and objective: Screening of drug activity is the starti-ng point for innovative drug research and the decisive step. With the pr-ogress of the discipline of molecular pharmacology, molecular biologyand cell biology, high-throughput screening has become one of the im-portant means of drug discovery. Using high-throughput screening mod-el can efficiently screen lead compounds in a short time, which then used for the development of new drugs. G protein-coupled receptors-as drug targets are most widely studied. To establish the screeningmodel according to the receptor ligand binding theory, can find thecompounds combination with G protein-coupled receptor.The cannabinoid receptor two is a subtype of cannabinoid rece-ptors.The genes for this receptor(CNR2) was shown to encode for360amino acid long,7-transmembrane G-protein coupled receptor.TheCB2receptor is mostly in peripheral tissue associated with immunefunctions, including macrophages and B cells. It is present in the cen-tral nervous system under physiological conditions, but the expression is very low. In addition, the uterus and gastrointestinal tract aslo havethe distribution of the CB2receptor. A growing body of evidence sug-gests that CB2receptor plays a critical regulatory role in the patholo-gyical process of many diseases.CB2agonists have been reported to-inhibit inflammation, improve microcirculation, anti-cancer, and analg-esic. Therefore, researching and development of drugs with the CB2activity, may be with the great medical value.The research aims to establish a dual luciferase reporter gene scree-ning model of the cannabinoid receptor two agonist. On the basis ofModel was successfully established,we preliminary screen a seriesof traditional Chinese medicine monomer and abstracts.In order toscreen the drugs with agonist activity for the development of newdrugs,at the same time the precious traditional Chinese medicine res-ources to be used more effectively.The first part: Construction and eukaryotic expression ofpIRES2-EGFP-CB2plasmidMethods: Full length DNA of CB2receptor genes amplified fr-o-m plasmid pcDNA3.1(+)-CB2was inserted into pIRES2-EGFP pla-smid by gene recombinantion technology. The recombinant plasmidwas transfected into HEK293cells. The expression of GFP was exa-minedunder fluorescence microscope and the expression of CB2wasexamined by RT-PCR and Western blot. Results: The specificity of PCR products by DNA sequencingand restriction endonuclease reactions demonstrated that recombinantpIRES2-EGFP-CB2plasmid was successfully constructed. Green fluor-escence expression of the HEK293cells can be observed under flur-escence microscope and high expression of CB2was detected byRT-PCR and Western blot.Conclusion: pIRES2-EGFP-CB2has been successfully constructedand expressed in eukaryotic cells.The second part: Establishment of cell line expression ofthe CB2receptor and dual luciferase reporter genesMethods: Plasmid pIRES2-EGFP-CB2, pGL4.29[luc2P/CRE/Hygr-o] and PRL-TK were cotransfected into HEK293cells in96wellsplate. The GFP positive cells were isolated by G418resistance scree-ning and flow cytometry sorting.The expression of GFP was exami-ned under fluorescence microscope and the expression of CB2wasexamined by RT-PCR and Western blot. Further to screen cell lineswhich stable expression of the dual-luciferase reporter genes by mea-surement the luciferase activity.Results: The expression of CB2receptor in stable expression celllines were detected by RT-PCR and Western blot and the green fluoresc-ence can be observed in the fluorescence microscope. Through detec-ting luciferase activity, the cell line with the highest activity of dual luciferase reporter gene was screened.Conclusions: Successfully constructed the stable expression of CB2and dual-luciferase reporter genes cell lines, laid the foundation forhigh-throughput screening of active substances.The part three: Investigation of screening model for CB2receptor agonist and drug screeningMethods:Optimizing the agonist concentration and incubation time.The screening system was assessed with regard to high-throughput、index,such as Z ’factor, signal to background and signal to noise,etal.In addition,using CB2antagonist AM630to verify the specificityof the model. On this basis, a series of traditional Chinese medicinemonomer and abstracts were screened by the high-throughput system.Results: The greatest relative induction rate can be achieved af-ter6h when giving1μmol/L of HU-308. Other indexes includedZ ’factor of0.84, signal to background of7.45and signal to noise of66.65,which represent good stability.Compared with control,the active-ties of luciferase reporter genes significantly increased when adminis-tered with AM630(P<0.001),which suggested that the cell modelkept good specificity and could be used as a screening model forCB2agnosit.With the screening model,Two kinds of Chinese medici-ne monomer showed higher potency for CB2receptor agonist activit-y. Conclusion:Through investigate the stability and specificity of th-is model,sugget it was stable and reliable.The screening results couldbe credible.