摘要
目的:构建和表达带蛋白转导结构域(protein transduction domain,PTD)的Foxp3(forkhead boxP3)PRD缺失突变体(PTD-ΔPRD)融合蛋白,并探讨其对脾混合淋巴细胞反应的影响。方法:应用PCR技术扩增获得小鼠ΔPRD片段核酸序列,将其分列插入到带PTD的pET28a-PTD和pET28a-PTD-eGFP原核表达载体中,并在大肠埃希菌Rosetta(DE3)中表达融合蛋白,利用镍柱获得并复性融合蛋白;Western印迹鉴定目的蛋白表达的正确性、流式细胞术和Western印迹检测融合蛋白穿膜进入小鼠T淋巴细胞瘤株EL-4细胞中的能力、双向混合淋巴细胞反应分析融合蛋白对T淋巴细胞增殖的影响。结果:融合蛋白可在大肠埃希菌Rosetta(DE3)中表达,并获得了大量纯化的融合蛋白。流式细胞术和Western印迹显示融合蛋白穿越细胞膜进入EL-4细胞核中,混合淋巴反应初步证明融合蛋白具有抑制T淋巴细胞增殖的功能。结论:成功表达小鼠PTD-ΔPRD Foxp3融合蛋白,该融合蛋白能有效进入细胞核内,抑制T淋巴细胞增殖。
Objective To express protein transduction domain（PTD）-deletion proline domain（ΔPRD）Foxp3 fusion protein,and to analyze its influence on mixed lymphocyte reaction in mice.Methods We cloned mouse ΔPRD of Foxp3 gene by PCR,and inserted it into pET28a-PTD,pET28a-PTD-eGFP vector,then expressed fusion proteins in E.coli Rosetta（DE3）.The fusion proteins were purified and refolded by Profinity IMAC Ni2＋-Charged Resin.The expression of fusion proteins was identified by Western blot.Flow cytometry assay was used to detect the effect of PTD-ΔPRD fusion protein to transduce into mouse EL-4 cells.The ability of fusion protein to inhibit the proliferation of EL-4 cells was analyzed by two-way mixed lymphocyte reaction.Results The PTD-ΔPRD fusion proteins were expressed and purified efficiently.Western blot and flow cytometry indicated that PTD-ΔPRD fusion protein was transduced into EL-4 efficiently.Mixed lympocyte reaction assay showed that PTD-ΔPRD fusion protein had the bioactivity to inhibit the proliferation of EL-4 cells.Conclusion The PTD-ΔPRD fusion protein was expressed in E.coli system and could be transduced into cells effectively,suggesting that PTD-ΔPRD fusion protein may be an inhibitor in lymphocytes from mouse spleen.
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2009年第12期1224-1230,共7页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(30671984)~~
