摘要
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。
Objective： To express and purify mouse PTD-Foxp3 fusion protein and investigate its biofunction in T ceils. 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 ceils 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.
出处
《江苏大学学报（医学版）》
CAS
2007年第3期188-192,196,共6页
Journal of Jiangsu University：Medicine Edition
基金
国家自然科学基金资助项目(30671984
30471627
30300169)
江苏省自然科学基金资助项目(BK2004405)
关键词
PTD
Foxp3融合蛋白
免疫抑制
protein transduction domain
PTD-Foxp3 fusion protein
immune inhibition
