Construction and Identification of a Yeast Two-hybrid Bait Vector for Screening of Homo Sapiens Peroxiredoxin II-binding Protein

Objective: To construct and identify a yeast two hybrid system bait vector for screening of homo sapiens PrxⅡbinding proteins,and to study its effect on the growth of yeast cells and the activation of reporter genes.Methods: Full fragment of ORF of peroxiredoxinⅡ cDNA was amplified using PCR and directly ligated to the pGBKT7 vector.Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.The plasmid was transformed into the yeast cell AH109,and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay.Results: The fragments of PrxⅡprotein were successfully obtained.The recombinant pGBKT7-PrxⅡplasmids and empty pGBKT7 vector could grow white colonies on SD/-Trp/X-α-gal plates and none could survive on SD/-His/-Trp/X-α-gal and SD/-Ade/-Trp/X-α-gal plates.After being cultured in SD/-Trp liquid medium for 16 h,the OD600 of them were both 0.9.Conclusion: The bait plasmid pGBKT7-PrxⅡ constructed expresses correctly,and can not activate the transcription of reporter gene alone.The yeast two hybrid GAL4 system3 can be utilized to fish PrxⅡregion interacting protein.