Construction and Expression of"Bait Plasmid"of Nucleolin in RNA-Protein Hybrid HunterTM System

【Objective】Using C23 gene to construct the bait vector,which can express C23 protein in yeast cell and interact with it,can be used in RNA-Protein Hybrid asbait plasmidto screen the mRNAs from the cDNA library.【Methods】PCR was performed to amplify the C23 gene from the vector pEGFP-N1-C23.The ORF of the C23 was inserted into thebait plasmid pYESTrp3 after digestion by the restricted endonuclease of EcoR Ⅰ and Not Ⅰ.After verified by sequencing,the plasmid was transformed into the yeast L40-ura3 / pHybLex/Zeo-MS2.The C23 protein expression in the yeast cell was confirmed by Western blot.Colony-lift filter assay was used to verify whether the constructed plasmid alone could activate the reporter gene in the yeast cell.【Results】Digested by two endonucleases,the recombined vector pYESTrp3-C23 produced anticipated fragment.Assayed by Western blot,it showed that the yeast cell transformed pYESTrp3-C23 vector had positive signal which could not be seen in the control.Tested by the colony-lift filter assay,both the pYESTrp3-C23 vector and control vector(pYESTrp3) could not activate LacZ reporter gene in the yeast,while the cell transformed with positive control plasmid pYESTrp3/IRP carrying whole GAL4 gene turned blue,that indicated the reporter gene was activated.【Conclusion】 Bait plasmid is constructed correctly and can express C23 proteins in the yeast cell but can not activate transcription of LacZ reporter gene alone.The plasmid can be used in Protein-RNA Hybrid.