Abstract:It is to establish HEK293 cell line which could stably express sweet taste receptor protein T1R2/T1R3.Firstly,extract total mRNA from mouse tongue tissue,then amplify Gα15,T1R2 and T1R3 target gene fragment by RT-PCR with self-designed primers and above total mRNA template.Then,establish the recombinant plasmid pEGFP-C1-Gα15,pDsRed1-N1-T1R2,pcDNATM6.2/N-YFP-DEST-T1R3 and introduce them into the HEK293 cells by liposome.After resistance screening,the HEK293 cell line with the ability of stable expressing T1R2/T1R3 was obtained in a limit dilution method.Finally,the stable cell line with sweet receptor protein T1R2/ T1R3 was identified by RT-PCR,fluorescence microscopy and Western blot.The results in gene and protein level show that Gα15、T1R2/T1R3 are successfully introduced into HEK293 cell line and are stably expressed.The establishment of HEK293 cell line provides a stable cell source for the study of sweetness mechanism in vitro(such as sweet recognition thermodynamics,etc.) at cell
