Abstract: This test was intended to establish a cell screening model for discovering new antagonists of chicken melanocortin-4 receptor (cMC4R) in vitro,and provide some basic research for screening ingredients which could interact with cMC4R and have activities from traditional Chinese medicine.The eukaryotic expression plasmid cMC4R-pcDNA3.1(+)-myc and a reporter gene plasmid pGL4.29[luc2p/CRE/Hygro] were co-transfected with the ratio of 1:5 into CHO-K1 cell line.Monoclone were obtained by limiting dilution method two weeks later.The obtained monoclone reacted with Forskolin and NDP-MSH for 6 h,respectively,choosing the monoclonal cell line which the relative expression of firefly luciferase was highest as a positive.Extract total RNA of positive cells,and detect the transcription of cMC4R gene and luciferase reporter gene by RT-PCR.To identify and optimize the assay condition,the effects of some factors were examined using NDP-MSH,including final concentration of NDP-MSH,incubation time,and final concentration of DMSO,and using Z'-factor to evaluate the screening method.The results showed that 5 monoclone (A2,F7,F8,F9 and H10)were obtained,while took A2 as the positive cell lines in which the relative expression of firefly luciferase was highest;The cMC4R gene (996 bp),luciferase reporter gene (1 653 bp) were detected in the A2 cell lines by RT-PCR;A stable cell line was established for cMC4R antagonist screening,the Z'-factor was 0.82 on the condition the final concentration of NDP-MSH was 10^-9 mol/L,the incubation time was 8 h and the final concentration of DMSO was less than 2%.The results showed that the established cell screening model could be used in the screening of cMC4R antagonists,and laid the foundation for extracting the effective substances from traditional Chinese medicine.

Key words: chicken melanocortin-4 receptor (cMC4R); reporter gene; cell screening model