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Expressed, Purified, Identified and Biological Fuction Analysised of Human FOXP3 Fusion Proteins

Author: TianYuXiang
Tutor: ShaoQiXiang
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: The PTD-hFOXP3 fusion protein Regulatory T cells Immunosuppressive
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 8
Quote: 0
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Abstract


Foxp3 is a method of inhibiting the transcription factor , is the relative specificity of regulatory T cells (regulatory Tcells, Treg) flag , and maintaining Treg the occurrence , development, function and plays an important role . Foxp3 gene mutation can lead to the Treg cell mediated immune tolerance defects . The study found that the expression of exogenous Foxp3 enables non- regulatory T cells with similar Treg cell-like phenotype and immunosuppressive function . HIV-1 (human immunodeficiency virus type 1) the gene encoding the trans-activating protein (transactivator, TAT) protein transduction domain (protein transductiondomain, PTD) be able to carry the exogenous protein through almost all eukaryotic membranes into the cytoplasm and nucleus , while efficient , fast , and does not affect the activity of the protein . Objective: To construct with PTD original expression vector pET28a - PTD - hFOXP3 , pET28a - PTD - eGFP - hFOXP3 , expression and purification of the PTD - hFOXP3 , PTD - eGFP - hFOXP3 fusion protein , study fusion protein to wear film transfer guide into the T-cell efficiency and changes the transduction protein before and after T cell activation stimuli . Methods : the use of molecular biology techniques to construct pET28a - PTD - hFOXP3 pET28a - PTD - eGFP - hFOXP3 prokaryotic expression vector , and expressed in Escherichia coli Rosetta (DE3) fusion protein was purified by Ni 2 separation column purification of the fusion protein. Western-blot detection of the correctness of the fusion protein , flow cytometry , Western-blot technology and laser confocal detection fusion protein through the cell membrane into the human T- lymphocyte tumor lines Jurkat cells ability to take advantage of the lymphocyte proliferation test detect its impact on the function of T cells . The result : the right to build a pET28a - PTD- hFOXP3 pET28a - PTD- eGFP - hFOXP3 fusion protein expression vector , expressed and purified PTD - hFOXP3 fusion protein PTD -eGFP - hFOXP3 . By flow cytometry , laser scanning confocal microscopy and western-blot analysis demonstrated that the fusion protein was able to efficiently into cells ; transduction the the PTD-hFOXP3 fusion proteins of Jurkat cells was significantly inhibited . Conclusion: The successful expression of biologically active PTD-hFOXP3, PTD-eGFP-hFOXP3 fusion protein to lay the foundation for further study of the PTD-hFOXP3 immune regulation function and its clinical application .

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