Foxp3突变体相关融合蛋白的表达、纯化、鉴定及生物学功能的初步研究

发布时间：2019-01-12 11:50

【摘要】： Foxp3是FOX(forkhead box)家族转录因子中的一员,是调节性T细胞(regulatory T cells,Treg)的特异性标志,同时对Treg的发生、发育、功能的维持与发挥等方面起着关键作用。研究发现表达外源性Foxp3可使非调节性T细胞具有类似调节性T细胞样的表型及免疫抑制功能。Foxp3的突变可能导致调节性的CD4~+CD25~+T细胞介导的免疫耐受缺陷。 HIV-1(human immunodeficiency virus type 1)基因编码的反式激活蛋白(transactivator,TAT)中的蛋白转导结构域(protein transductiondomain,PTD)能够携带外源性蛋白穿入几乎所有的真核细胞膜而进入细胞浆和细胞核内,同时具有高效、快速的特点。目的:构建带HIV-1穿膜序列原核表达载体pET28a-PTD-△Foxp3、pET28a-PTD-eGFP-△Foxp3、pET28a-PTD-eGFP-Foxp3,表达并纯化小鼠PTD-△Foxp3、PTD-eGFP-△Foxp3、PTD-eGFP-Foxp3融合蛋白,研究融合蛋白导入细胞的效率及其对T细胞活化增殖的免疫调节功能的影响。方法:利用基因重组技术构建pET28a-PTD-△Foxp3、pET28a-PTD-eGFP-△Foxp3、pET28a-PTD-eGFP-Foxp3原核表达载体,并在大肠埃希菌Rosetta(DE3)中表达融合蛋白,经Ni~(2+)分离柱纯化融合蛋白。Western-blot技术检测融合蛋白表达的正确性,流式细胞术检测融合蛋白穿越细胞膜进入小鼠T淋巴细胞瘤株EL-4细胞中的能力,并通过混合淋巴细胞反应初步分析融合蛋白对T细胞活化增殖的影响。结果:成功构建了pET28a-PTD-△Foxp3、pET28a-PTD-eGFP-△Foxp3、pET28a-PTD-eGFP-Foxp3融合蛋白表达载体,表达并纯化了PTD-△Foxp3、PTD-eGFP-△Foxp3、PTD-eGFP-Foxp3融合蛋白。通过流式细胞术分析证实表达的融合蛋白均能高效的转入细胞内;同时经混合淋巴细胞反应初步表明PTD-eGFP-Foxp3融合蛋白能明显抑制T淋巴细胞的活化增殖能力,而PTD-△Foxp3、PTD-eGFP-△Foxp3突变体融合蛋白抑制T细胞活化增殖的能力丧失。结论:成功表达具有生物学活性的PTD-△Foxp3、PTD-eGFP-△Foxp3、PTD-eGFP-Foxp3融合蛋白,为进一步更好地研究Foxp3的免疫抑制功能和构建表达人PTD-Foxp3相关融合蛋白奠定了基础。
[Abstract]:Foxp3 is a member of FOX (forkhead box) family transcription factors and a specific marker of regulatory T cell (regulatory T cells,Treg. It also plays a key role in the genesis, development, function maintenance and development of Treg. It has been found that the expression of exogenous Foxp3 can make non-regulatory T cells have a regulatory T-cell-like phenotype and immunosuppressive function. The mutation of Foxp3 may lead to regulatory CD4~ CD25~ T cell-mediated immune tolerance defects. HIV-1 (human immunodeficiency virus type 1) the protein transduction domain (protein transductiondomain,PTD) of the transactivator protein (transactivator,TAT) encoded by the gene can carry foreign proteins into almost all eukaryotic membranes and into the cytoplasm and nucleus. At the same time, it has the characteristics of high efficiency and fast. Objective: to construct a prokaryotic expression vector pET28a-PTD- Foxp3,pET28a-PTD-eGFP- Foxp3,pET28a-PTD-eGFP-Foxp3, with HIV-1 transmembrane sequence and purify mouse PTD- Foxp3,PTD-eGFP- Foxp3,PTD-eGFP-Foxp3 fusion protein. To study the effect of fusion protein on T cell activation and proliferation. Methods: the prokaryotic expression vector of pET28a-PTD- Foxp3,pET28a-PTD-eGFP- Foxp3,pET28a-PTD-eGFP-Foxp3 was constructed by gene recombination technique, and the fusion protein was expressed in Escherichia coli Rosetta (DE3). The fusion protein was purified by Ni~ (2) column. The expression of fusion protein was detected by Western-blot, and the ability of fusion protein to cross cell membrane into mouse T lymphocyte tumor EL-4 cell line was detected by flow cytometry. The effect of fusion protein on T cell activation and proliferation was analyzed by mixed lymphocyte reaction. Results: pET28a-PTD- Foxp3,pET28a-PTD-eGFP- Foxp3,pET28a-PTD-eGFP-Foxp3 fusion protein expression vector was successfully constructed, and PTD- Foxp3,PTD-eGFP- Foxp3,PTD-eGFP-Foxp3 fusion protein was expressed and purified. Flow cytometry showed that the expressed fusion protein could be efficiently transferred into the cells. At the same time, the mixed lymphocyte reaction showed that PTD-eGFP-Foxp3 fusion protein could significantly inhibit the activation and proliferation of T lymphocytes, while the PTD- Foxp3,PTD-eGFP- Foxp3 fusion protein could inhibit the activation and proliferation of T cells. Conclusion: the successful expression of PTD- Foxp3,PTD-eGFP- Foxp3,PTD-eGFP-Foxp3 fusion protein with biological activity lays a foundation for the further study of the immunosuppressive function of Foxp3 and the construction of human PTD-Foxp3 related fusion protein.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341

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