Screening For Candidate Molecular Interact With HBex1 By Yeast Two Hybrid System

Imatinib is the first-line drug in clinical treatment of Gastrointestinal Stromal Tumors(GIST) and Chronic Myelogenous Leukemia(CML). Many patient condition progress rapidly because of the development of secondary drug resistance to Imatinib, although they have obtained certain time of remission. Bex is a class of small molecular mediator in signal transduction, and its function has not fully understood. In previous study, we found hBexl involved in the process of Imatinib resistance. To clarify hBexl mediated signal transduction pathways is of great significance, for a clear mechanism of resistance to Imatinib and further development of molecular targeted drugs.Objective:To establish yeast two-hybrid system, screen hBex1 interacting proteins from K562 cells cDNA library, and analysis candidate proteins preliminarily.Methods:1. hBexl coding sequences originate from pEGFP-C1-hBexl plasmid was inserted into the pGBKT7 vector in proper orientation by gene engineering. And the constructed plasmid pGBKT7-hBexl was transformed into competent AH 109 S.cerevisiae, then toxicity effect and autonomous activation of pGBKT7-hBexl were testified by the phenotype assay.2. Total RNA was extracted from K562 cells, and ds cDNA was reversed through the SMART technology and amplified by LD-PCR technology.3. pGBKT7-hBex1,pGADT7-Rec,ds cDNA were co-transformed into competent AH 109 S.cerevisiae, and positive clones were isolated by solid dropout SD/-Ade/-His/-Leu/-Trp culture media.4. Bait plasmid and target plasmid extracted from positive clones were co-transformed into competent AH109 S.cerevisiae to testify positive protein-protein interaction by solid dropout SD/-Ade/-His/-Leu/-Trp culture media again.5. Plasmids extracted from positive target clones were sequenced and analysised in bioinformatics.Results:1.The yeast bait expression plasmid pGBKT7-hBexl was constructed and identified by restriction enzyme digesting and DNA sequencing. pGBKT7-hBex1 was nontoxic and no self-activation to AH 109 S.cerevisiae.2. There are seventeen Ade+/His+/Leu+/Trp~+positive clones were isolated through sereening the K562 ds-cDNA library with pGBKT7-hBex1 as bait plasmid by co-transformed into competent AH 109 S.cerevisiae, then thirteen clones were obtained by retesting phenotype in solid dropout SD/-Ade/-His/-Leu/-Trp culture media.3. Thirteen plasmids extracted from positive clones and isolated by Amp~+-LB culture media were co-transformed into competent AH109 S.cerevisiae with pGBKT7-hBex1 respectively, and AH109 S.cerevisiae co-transformed of both plasmids can grow in solid dropout SD/-Ade/-His/-Leu/-Trp culture media.4. Five different candidate genes were obtained by plasmids inserts sequencing and bioinformatic analysis.Conclusions:1.The yeast bait expression plasmid pGBKT7-hBexl was constructed and suitable for yeast two hybrid screening.2. Five different candidate genes were obtained through sereening the K562 ds-cDNA library by yeast two hybrid technology.