中国生物工程杂志

Objective: To construct the BD bait vector in yeast two-hybrid system by using pBK-CMV-HA-EBP50 plasmid. Methods: Coding region was obtained from the EBP50 plasmid by PCR. EcoR I/ BamH I -digested PCR products and bait vector pGBKT7 were ligated, and then transformed into E.coli DH5α to obtain the transformants. The positive clones were screened and identified by restriction endonuclease analysis and DNA sequencing. The correct recombinant plasmid pGBKT7- EBP50 was transformed into the competent yeast AH109. The activation of pGBKT7- EBP50 on the reporter genes and its toxicity on AH109 were also determined. Results: The size of the inserted fragment and recombinant plasmid was consistent with theoretically predicted value. There were no activation of pGBKT7- EBP50 on the reporter genes and no toxicity on AH109. Conclusion: The bait plasmid pGBKT7- EBP50 is successfully constructed for the yeast two-hybrid system., may be used to screen for the novel protein-protein interactions and further contribute to the study of EBP50 roles in regulation of cellular functional activities.