PTD-Foxp3融合蛋白的表达、纯化及其生物学功能初步研究

目的:构建带HIV-1 TAT转录因子的穿膜序列(Protein transduction domain,PTD)的pET28a-PTD-Foxp3原核表达载体,表达纯化PTD-Foxp3融合蛋白,并研究其生物学作用.方法:利用基因重组技术构建pET28a-PTD-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Ni2 分离柱纯化pET28a-PTD-Foxp3融合蛋白.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抑制T细胞活化增殖的生物学作用.结果:成功地构建了pET28a-PTD-Foxp3融合蛋白表达载体,表达并纯化了pET28a-PTD-Foxp3融合蛋白.通过流式细胞术分析证实pET28a-PTD-Foxp3融合蛋白能有效地进入细胞内;同时经混合淋巴细胞反应证明该融合蛋白能明显抑制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融合蛋白免疫抑制功能和构建表达人PTD-Foxp3融合蛋白,最终应用于临床疾病的治疗奠定了基础.

Expression, purification of PTD-Foxp3 fusion protein and analyse its bioactivity

Objective: To express and purify mouse PTD-Foxp3 fusion protein and investigate its bio-function in T cells.Methods: Expression vector of PTD-Foxp3 was constructed with inserting the PCR products of PTD-Foxp3 into pET28a Vector.The fusion protein was expressed by E.coli Rosetta(DE3) and purified by Bio-Rad Profinity IMAC Ni-Charged Resin.A flow cytometry assay was used to detect the effect of PTD-Foxp3 to transduce into the cell cytoplasm.The capability of PTD-Foxp3 for inhibiting the proliferation of T cells was analyzed by mixed lympocyte reaction(MLR). Results: A prokaryotic expression vector of pET28a-PTD-Foxp3 was successfully constructed and the fusion protein was expressed and purified efficiently.The results of flow cytometry indicated that pET28a-PTD-Foxp3 fusion protein could transduce into EL-4 efficiently.MLR confirmed that the fusion protein could inhibit the proliferation of T cells. Conclusion: The present study describes PTD-Foxp3 fusion proteins as a powerful anti-T cell proliferation tools.This study suggested that the PTD-Foxp3 fusion protein may be use for clinical autoimmune disease and transplantation rejection therapy.