Establishment of a Chinese Hamster Ovary Cell Line Stably Expressing Nicotinic Acid Receptor GPR109A

Aim To construct pEGFP-GPR109A recombinant plasmid by inserting GPR109A gene into pEGFP-N3 vector and establish a Chinese hamster ovary(CHO)cell line stably expressing nicotinic acid receptor GPR109A. Methods The pEGFP-GPR109A recombinant plasmid was constructed,which was subsequently transformed into DH5α E coli.After identification by PCR,digestion with restriction endonuclease and sequencing,the recombinant plasmid was transfected into CHO cells via lipofectamine 2000.The stable transfectants were screened by antibiotic G418.The fluorescent signal of cloned cell lines were detected by fluorescent microscope.The GPR109A gene mRNA expression was analyzed by RT-PCR and the fusion protein expression of green fluorescence protein and nicotinic acid receptor GPR109A(GFP-GPR109A) was detected by Western Blotting.The cell localization of fusion protein was detected by laser scanning confocal microscope. Results The results of PCR,restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pEGFP-GPR109A.Western Blotting showed that the fusion protein GFP-GPR109A was expressed stably in CHO cells.The fusion protein was mainly localized on cell membrane detected by laser scanning confocal microscope. Conclusion The eukaryotic expression vector pEGFP-GPR109A has been successfully constructed and a CHO cell line stably expressing fusion protein GFP-GPR109A was established,which facilitated the physiological and pathological function research of GPR109A and also provided important foundation for following up drug screening for atherosclerosis treatment.