Screening For Proteins Interacting With DND1 By Yeast Two-Hybrid System

Objective : To have an insight into the function of Dnd1 and lay a foundation for investigating the signal pathway as well as the role of Dnd1 gene in the development of testicular germ cell tumors, yeast two-hybrid system was used to screen proteins interacting with DND1.Methods:①The cDNA fragment of Dnd1, which was amplified from mouse embryo cDNA library by PCR, was inserted into the bait vector pDBLeu. Identifying the recombinant plasmid by restriction endonuclease analysis and PCR assay.②The recombinant plasmid pDBLeu-Dnd1 was transformed into yeast strain MaV203. The BD-Bait protein was examined for the reporter gene LacZ activation byβ-galactosidase filter assay and toxicity to the yeast strain through examining the proliferation speed.③The AD-Library plasmid was transformed into yeasts containing the bait plasmid. The yeasts containing the bait and prey plasmid were plated on synthetic dropout nutrient medium. The screen of positive clones was performed for three times via analyzing the type of LacZ byβ-galactosidase filter assay.④The library plasmids in positive clones were isolated and then transformed into electrocompetent cells of E.coliDH10B in order to obtain transformants containing AD-Library. The prey AD-Library plasmids were extracted from DH10B cells and were transformed into yeast strain MaV203 containing pDBLeu-Dnd1 to retest the interactions between proteins. After restriction endonuclease and PCR assay, the Library plasmids in authentic positive clones were sequenced and analysed by bioinformatic techniques.Results :①The orientation of inserted cDNA fragment and the ORF of Dnd1 were confirmed correctly in combinant plasmid pDBLeu-Dnd1 by restriction endonuclease analysis and sequencing.②The fusion protein had no self-activation and toxicity to the host strain.③172 positive clones were obtained by the first round of screening the mouse embryo cDNA library, 10 positive clones in the second round and 8 positive clones were confirmed to be authentic positive in the third.④Sequnced and compared the 8 sequnces with the sequnces in the GenBank, we found 4 proteins interacted with DND1. They were Jun oncogene, Actinin alpha 1, Nuclear Receptor Binding Protein 1 and Chloride Channel 2.Conclusions:①The bait plasmid pDBLeu-Dnd1 was succesfully constructed.②The bait plasmid pDBLeu-Dnd1 can be used to screen the mouse embryo cDNA library.③DND1 interactive factors obtained by screening the mouse embryo cDNA library were Jun protein, Actinin alpha 1, Nuclear Receptor Binding Protein 1 and Chloride Channel 2.