Expression and construction of "bait plasmid" containing human GR ligand-binding domain in yeast two-hybrid system / 第三军医大学学报

ABSTRACT

Objective To construct a bait vector containing human glucocorticoid receptor(GR) ligand binding domain (LBD) in yeast two hybrid system in order to screen its cDNA library Methods PCR was used to amplify GR LBD fragment from the fetal liver cDNA library with the primers designed in accordance with the sequence in GenBank GR LBD The product was inserted into pGEM(r) T Vector Systems After verified with restriction endonuclease digestion of SmaⅠ/SalⅡ, the vector was inserted into the "bait plasmid" pGBKT7 (named as pGBKT7 GR LBD) After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and the transformants were selected on SD/ Trp plates The interaction between GR and SRC 1 was tested by ? gal activity with yeast two hybrid system Results The amplified product of 893 bp was inserted into pGEM(r) T Vector and proven to be successful with double restriction enzyme digestion Sequence analysis revealed that the sequence was correctly inserted into pGBKT7 with a right reading frame AH109 [pGBKT7 GR LBD] grew on SD/ Trp plates, but not on the other selective media GR could interact with SRC 1 Conclusion The bait plasmid pGBKT7 GR LBD constructed expresses GR LBD correctly, and can't activate the transcription of reporter gene alone in yeast two hybrid system