PTD-Foxp3融合蛋白的表达纯化和生物学功能的初步研究

发布时间：2018-03-16 06:33

本文选题：PTD　切入点：Foxp3融合蛋白　出处：《江苏大学》2007年硕士论文　论文类型：学位论文

【摘要】： Foxp3属于FOX(forkhead box)转录因子家族，对调节性T细胞(regulatory T cells Treg)的发生及生物学功能的发挥具有十分重要的作用。研究发现表达外源性Foxp3可使非调节性T细胞获得调节性T细胞的表型及功能。蛋白转导结构域(protein transduction domain,PID)是HIV-1(human immunodeficiency virus type 1)基因编码的反式激活蛋白(Trans-activator transcription，TAT)中的一段具有穿膜功能的多肽，能够携带外源性蛋白穿过几乎所有的真核细胞膜进入细胞浆和核内。目的：构建带HIV-1 PTD序列的原核表达载体pET28a-PID，表达小鼠PTD-Foxp3融合蛋白，并研究其对T细胞免疫功能的调节作用。方法：利用基因重组技术构建pET28a-PTD原核表达载体及pET28a-PID-Foxp3融合蛋白表达载体，并在大肠埃希菌Rosetta(DE3)中表达融合蛋白，经Ni~(2+)分离柱纯化带His标签的PID-Foxp3融合蛋白。采用Western-blot技术鉴定所表达的PTD-Foxp3蛋白；运用流式细胞术确认融合蛋白穿膜功能；通过混合淋巴细胞反应初步分析融合蛋白对T细胞活化增殖能力的调节。结果：成功地构建了pET28a-PID-Foxp3融合蛋白表达载体，，表达并纯化了PTD-Foxp3融合蛋白。通过流式细胞术分析证实PTD-Foxp3融合蛋白能有效的进入细胞内；而且能在混合淋巴细胞反应中有效抑制T细胞的活化增殖能力。结论：成功表达具有生物学活性的PTD-Foxp3融合蛋白，为进一步研究PTD-Foxp3融合蛋白生物学功能和构建表达人PTD-Foxp3融合蛋白奠定了基础。
[Abstract]:Foxp3 belongs to the FOX(forkhead box) transcription factor family, It is found that the expression of exogenous Foxp3 can induce the phenotype and function of regulatory T cells. Protein transduction domain (transduction) is a transmembrane transcriptional peptide encoded by HIV-1(human immunodeficiency virus type 1 (HIV-1(human immunodeficiency virus type 1), which can carry foreign proteins through almost all eukaryotic cell membranes into the cytoplasm and nucleus. Aim: to construct a prokaryotic expression vector pET28a-PIDwith HIV-1 PTD sequence to express mouse PTD-Foxp3 fusion protein and to study its regulatory effect on T cell immune function. Methods: pET28a-PTD prokaryotic expression vector and pET28a-PID-Foxp3 fusion protein expression vector were constructed by gene recombination technique, and the fusion protein was expressed in Escherichia coli Rosetta-DE3. PID-Foxp3 fusion protein labeled with His was isolated and purified by Ni~(2 column. The expressed PTD-Foxp3 protein was identified by Western-blot technique, and the transmembrane function of the fusion protein was confirmed by flow cytometry. The regulation of fusion protein on T cell activation and proliferation was preliminarily analyzed by mixed lymphocyte reaction. Results: pET28a-PID-Foxp3 fusion protein expression vector was successfully constructed and PTD-Foxp3 fusion protein was expressed and purified. Flow cytometry showed that PTD-Foxp3 fusion protein could effectively enter the cells. Moreover, it can effectively inhibit the activation and proliferation of T cells in mixed lymphocyte reaction. Conclusion: the successful expression of PTD-Foxp3 fusion protein with biological activity lays a foundation for further study of biological function of PTD-Foxp3 fusion protein and construction of human PTD-Foxp3 fusion protein.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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