CAJ | 학술논문

目的:构建和表达带蛋白转导结构域(protein transduction domain,PTD)的Foxp3(forkhead box P3)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.
목적:구건화표체대단백전도결구역(protein transduction domain,PTD)적Foxp3(forkhead box P3)PRD결실돌변체(PTD-ΔPRD)융합단백,병탐토기대비혼합림파세포반응적영향.방법:응용PCR기술확증획득소서ΔPRD편단핵산서렬,장기분렬삽입도대PTD적pET28a-PTD화pET28a-PTD-eGFP원핵표체재체중,병재대장애희균Rosetta(DE3)중표체융합단백,이용얼주획득병복성융합단백;Western인적감정목적단백표체적정학성、류식세포술화Western인적검측융합단백천막진입소서T림파세포류주EL-4세포중적능력、쌍향혼합림파세포반응분석융합단백대T림파세포증식적영향.결과:융합단백가재대장애희균Rosetta(DE3)중표체,병획득료대량순화적융합단백.류식세포술화Western인적현시융합단백천월세포막진입EL-4세포핵중,혼합림파반응초보증명융합단백구유억제T림파세포증식적공능.결론:성공표체소서PTD-ΔPRD Foxp3융합단백,해융합단백능유효진입세포핵내,억제T림파세포증식.
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 Ni~(2+)-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.