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Screening of ssDNA Aptamers Specific to Catalytic Region of Glucosyltransferase by Liquid-SELEX

Author: LiHui
Tutor: WangMingGang
School: Lanzhou University of Technology
Course: Biochemistry and Molecular Biology
Keywords: The glucosyltransferase catalytic District Liquid -SELEX Random ssDNA library Aptamers Gel retardation
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 116
Quote: 1
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Abstract


Glucosyltransferase (Glucosyltransferase, GTF) is important cariogenic factors of Streptococcus mutans , catalytic sucrose synthetase glucan , including a variety of extracellular polysaccharides . GTF structure comprising a catalytic region (Catalytic, CAT) and glucan -binding region (Glucan binding, GB , or GLU) two functional sections , wherein the catalytic region (CAT) is an important functional segment . First, using genetic engineering methods in E. coli , were induced to express protein with NusA rNusA-CAT fusion protein and NusA control . Bacterial After sonication , the recombinant protein exists in the sonicating supernatant . Use of C-terminal His tag carrier and a metal chelate chromatographic purification of recombinant proteins . Anthrone - sulfuric acid method proved to have good catalytic activity of recombinant rNusA-CAT protein . The traditional SELEX technology screening target molecules , you first need to target molecules purified and fixed , and thus to the native conformation exists in the liquid phase in the non- purified target proteins can not be screened by the traditional method . To solve this problem , the study of the basic principles of the gel retardation (EMSA) introduced subtractive SELEX screening process , the initial establishment of a new screening method based gel retardation , non - purified target protein liquid phase screening (liquid -SELEX technology). Experiments using random region is 35 bases , ssDNA library of full length 78 NT to express rNusA-CAT protein the unpurified supernatant for the purpose of target , in order to express the NusA protein unpurified supernatant abatement target , using liquid -SELEX the technology for the screening of ten . Radioisotope detection : With the increase in screening rounds , ssDNA library enriched gradually enriched ssDNA ligand specific recognition rNusA-CAT protein , without identifying NusA protein . The results achieved to lay the foundation for the future function of GTF inhibitors obtained and the prevention and control of dental caries . Liquid -SELEX technology to establish non- purified natural conformation of protein screening patient serum , saliva , bodily fluids , such as in the future as a screening targets fast , effective serum or body fluids of disease markers and molecular probes a new technology to bring new ideas to the diagnosis and treatment of diseases .

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria
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