Construction and Self-Activation Detection of Bait Vector Containing of Mouse Cytomegalovirus Immediate-Early Gene M122 in Yeast Two-Hybrid System

Objective To construct a bait vector containing mouse cytomegalovirus(MCMV) M122 gene in yeast two-hybrid system and detect the self-activation of this recombinated plasmid in order to screen the cDNA library of mouse fetal brain.Methods The DNA fragment of coding M122 was amplified by reverse transcriptase polymerase chain reaction,and then inserted into pMD18-T simple vector.After verified with restriction endonuclease digestion of EcoRⅠand SalⅠ,the right fragment of M122 gene determined by sequencing was subcloned into pGBKT7-BD vector.Then right fragment of recombinant plasmid pGBKT7-M122 detected by both the same endonuclease and sequence analysis was transformed into the yeast cell AH109.The transformed yeast cells were plated on nutrient deficiency medium SD/-Trp and SD/-Trp containing X-α-Gal.Positive plasmid pCL and empty pGBKT7 vector as controls were also transformed into the yeast strain AH109,and the transformed yeast cells were respectively plated on nutrient deficiency medium SD/-Trp and SD/-Leu containing X-α-Gal.The color,number and size of colonies on the plates were observed.Results The pGBKT7-M122 plasmid was successfully constructed.The colonies containing pGBKT7-M122 were white,while the colonies of the positive control were blue.The colonies containing pGBKT7-M122 and empty pGBKT7 vector were similar on the number and size.Conclusion The recombinated plasmid pGBKT7-M122 had no autoactivation and toxicity,and was proved to be suitable for the further research on the interactors with M122 in the yeast two-hybrid system.