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The Preparation and Research for Bio-Founction Analyse of the PTD-△NFATminiDBD Fusion Protein

Author: PengZuo
Tutor: ShaoQiXiang
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: The prokaryotic expression PTD NFAT Immunosuppressive
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 8
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Abstract


Objective: The Clone △ NFATminiDBD gene construct prokaryotic expression vector with PTD transmembrane sequence , in vitro expression and purification of the fusion protein refolding , a preliminary study of its biological function . Method: by overlap PCR construct containing the mutation site of R466A/T533G two people NFAT amino acids 391 to 583 minimum DNA binding sequence that △ NFATminiDBD , and cloned into the pMD18-T vector , double digestion and sequence analysis . The sequencing of the right of the people △ NFATminiDBD gene was cloned into with wear film sequence of pQE30a - the PTD pQE30a - PTD - eGFP original nuclear expression vectors and not with wear film sequence of pQE30a original expression vector , and the engineered bacteria E · Coli M15 expression , 6 × His tag - containing fusion protein; nickel column purification of the fusion protein using the Bio-Rad Company , and protein refolding . Correctness of fusion protein expression using Western-blot detection , flow cytometry and Western-blot detection fusion protein to enter the ability of the human T- lymphocyte tumor lines Jurkat cells , T cell proliferation inhibition test preliminary analysis of the fusion protein on T cell proliferation the adjustment of the capacity . Results : the successful building of not having to wear membrane function of pQE30a - △ NFATminiDBD prokaryotic expression vector , and wearing the membrane function of pQE30a - the PTD - △ NFATminiDBD pQE30a - PTD - eGFP pQE30a - PTD - △ NFATminiDBD - eGFP prokaryotic expression vector , the expression purification and refolding of the fusion protein with a 6 × His tag . Western-blot confirmed the accuracy of the fusion protein expression , flow cytometry and Western - blot analysis confirmed that The PTD can carry the fusion protein into the cell , T cell proliferation inhibition test proved that the fusion protein can significantly inhibit T cell activation and proliferation capacity Conclusion: successful expression of biologically active and has the function of transmembrane fusion protein PTD- △ NFATminiDBD preliminary experimental studies have shown that this fusion protein into the nucleus , significantly inhibited the proliferation of T-cells , to further study the immunomodulatory mechanism laid the foundation for future clinical applications .

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