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Study on the Clone and Biological Function Assay of Foxp3-△PRD/△FKH

Author: ShenWeiHong
Tutor: ShaoQiXiang
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: PTD Foxp3 △ PRD △ FKH Immunosuppressive
CLC: R346
Type: Master's thesis
Year: 2008
Downloads: 4
Quote: 0
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Abstract


Objective: To clone mouse Foxp3-△ PRD (N end of the proline-rich region missing the Foxp3 fragment ) and of Foxp3 -△ FKH ( C end FKH area missing the Foxp3 fragment ) gene construct with a transmembrane sequence PTD ( protein transduction domain ) prokaryotic expression vector , expression, purification and refolding of PTD- △ PRD the PTD- delta FKH , PTD -eGFP -△ PRD PTD -eGFP - △ FKH fusion protein , and explore its biological function , for further study Foxp3 the immunosuppressive mechanisms lay the foundation . Methods : ( 1 ) using PCR amplification mice △ PRD , and △ FKH gene was cloned into pMD18-T vector , single , double digestion and sequence analysis . ( 2 ) the correct sequencing the △ PRD and △ FKH cloned into with a transmembrane sequence pET28a-PTD and pET28a-PTD-eGFP prokaryotic expression vector , and expressed in Escherichia coli Rosetta (DE3) , obtained with His tag fusion protein , the fusion protein was purified by nickel column using Bio-Rad's protein refolding . (3 ) the use of the correctness of fusion protein expression detected by Western-blot , flow cytometry and Western-blot detection fusion protein penetrating into the mouse T lymphocytes the ability of the tumor strain EL-4 cells , and by mixed lymphocyte the reaction preliminary analysis of the regulation of the fusion protein on T cell activation and proliferation capacity . Results: successfully constructed prokaryotic expression vector with a penetrating expression , purification and refolding of His-tagged fusion protein , Western-blot confirmed the accuracy of the fusion protein , flow cytometry and Western-blot confirmed PTD can carry the Foxp3 protein into the cell , and mixed lymphocyte reaction proved the PTD-△ PRD fusion protein inhibits T-cell activation and proliferation capacity than the PTD-△ FKH fusion protein , indicating that the the FKH area in the C-terminal Foxp3 inhibition play an important role in the function , while the N-terminal proline-rich region also has a certain suppression function . Conclusion: The successful expression of Foxp3 related with biologically active transmembrane sequence fusion protein , Western-blot and flow cytometry confirmed that the fusion protein into cells and localized in the nucleus , for further study of the functional fragment of Foxp3 the immunosuppressive mechanisms laid the foundation .

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