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Screening for Candidate Molecular Interact with hBex1 by Yeast Two Hybrid System

Author: XiaoQian
Tutor: DingKeFeng
School: Zhejiang University
Course: Clinical
Keywords: Secondary resistance hBex1 Yeast two-hybrid
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 86
Quote: 0
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Abstract


Imatinib (Imatinib) is a clinical treatment of gastrointestinal stromal tumors (Gastrointestinal Stromal Tumors, GIST) and chronic myeloid leukemia (Chronic Myelogenous Leukemia, CML) in first-line drugs, but some patients in obtaining remission after a certain time, Imatinib produced due to secondary resistance, leading to disease progress rapidly. Bex is a class of small molecules that function has not been clarified information transduction intermediaries, in a previous study, we found hBex1 involved in the process of drug resistance to Imatinib. Clarify hBex1 mediated signal transduction pathways for explicit Imatinib resistance mechanisms, for the further development of molecular targeted drugs is of great significance. Objective: To establish the yeast two-hybrid system, from K562 cell cDNA library hBex1 interacting proteins, and preliminary identification of candidate proteins. Methods: From the pEGFP-C1-hBex1 will hBex1 gene open reading frame was cloned into the pGBKT7 vector to construct a recombinant plasmid pGBKT7-hBex1 and detect toxic to yeast plasmid and self-activating activity; 2 RNA was extracted from K562 cells by SMART technology and LD-PCR amplified K562 cells ds cDNA; 3 will pGBKT7-hBex1, pGADT7-Rec. ds cDNA of transform competent yeast, through SD /-Ade /-His /-Leu /-Trp plate screening positive clones; 4 will get positive clones pGADT7-library plasmid pGBKT7-hBexl competent yeast plasmids by SD /-Ade /-His /-Leu /-Trp plates pair interaction verification; 5. pair authentication After plasmid clones were sequenced and bioinformatics analysis. Results: 1. Constructed yeast expression plasmid pGBKT7-hBex1, testing confirmed that the recombinant plasmid in yeast cells without toxic effects, no self-activating activity; 2. Were transformed into yeast and from the SD /-Ade /-His /-Leu /-Trp plates 17 positive clones were screened out, crossed turn kind of tablet duplicate detection screening 13 positive clones; 3.13 clones extracted pGADT7-library with pGBKT7-hBexl recombinant plasmids were transformed competent yeast, can in SD / - Ade /-His /-Leu /-Trp plates growth; 4 pairs validated plasmid clones sequenced and bioinformatics analysis were obtained five candidate genes. Conclusions: 1. Construct pGBKT7-hBex1 recombinant plasmid, the plasmid suitable for yeast two-hybrid system; 2. Yeast two-hybrid system, from K562 cell cDNA library was screened five genes, which encode proteins interacted with hBex1.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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