Screening For Proteins Interacting With Human DND1by Yeast Two-Hybrid System

Objective:The objective of this research is to screen out proteins interaction with DND1from human brain cDNA library applying with the yeast two-hybrid technology and to analyze the functions of selected proteins. It makes the basis for further understanding the Dndl biological function and its signal pathway.Method:(1) Amplify DND1target fragmentusing human dnd1cDNA as a template by PCR. The target Dndl fragment was inserted to the bait vector pDBLeu to make the yeast two-hybrid bait plasmid pDBleu-dndl, and And identify the recombinant plasmid by restriction analysis and sequencing, also examinatethe pDBleu-dndl toxicity and self-activating. Thus it provides foundation for the next step to screen DND1interacting proteins;(2) Use yeast two-hybrid screening technology to select proteins which interact with DND1in normal human brain cDNA library,and positive clones were digested and sequenced;(3)CO transformate the bait plasmid pDBleu-dndl andpositive clone plasmids into the yeast AH109for further verification;(4) After rotary verification, the positive clone plasmids DNA were were sequenced and analysed by bioinformation techniques.Result:(1) Counstruct a shuttle plasmid pDBleu-dndl, and it is confirmed that the recombinant plasmid was non-toxic to yeast and non-self-activateion in yeast;(2) Identify four proteins which are GRN, FLAD1,EFEMP1, RNF3linteractwith human DND1with yeast two-hybrid system and bioinformation techniques.Conclusion:(1) The yeast two-hybrid bait plasmid pDBleu-dndlfor screenning human Dndl interacting protein was succesfully constructed; (2) We succesfully screened out four DND1interacting proteins using yeast two-hybrid technology, they are GRN,FLAD1, EFEMP1, RNF31.