Tat融合蛋白表达载体TAT-c-Myc的克隆化及蛋白表达研究

发布时间：2018-01-20 08:51

本文关键词： 原癌基因蛋白质c-myc 多能干细胞蛋白转导结构域载体构建　出处：《中华临床医师杂志(电子版)》2015年05期 　论文类型：期刊论文

【摘要】：目的构建重组表达载体TAT-c-Myc,在E.coli BL21中高效表达并纯化融合蛋白。方法经PCR获得编码人c-Myc的全基因序列,含有设计的限制性酶切位点的产物连接到含有TAT转导结构的原核表达载体PET-28b-TAT-V2上,得到重组表达载体TAT-c-Myc,转化大肠杆菌,应用IPTG诱导TAT-c-Myc融合蛋白的表达。表达产物用SDS-PAGE鉴定,亲和层析柱纯化,并应用Western blot检测蛋白的特异性,融合蛋白转导人皮肤成纤维(HSF)细胞的效果应用免疫荧光检测。结果成功构建了TAT-c-Myc融合蛋白的原核表达载体,在诱导下获得了高效表达。Western blot检测提示融合蛋白有良好的特异性。免疫荧光检测提示融合蛋白具有快速转导入HSF细胞内的能力。结论为进一步的通过蛋白转导方式诱导多能干细胞提供了物质基础。
[Abstract]:Objective to construct the recombinant expression vector TAT-c-Myc, express and purify the fusion protein in E.coli BL21. Methods the whole gene sequence of human c-Myc was obtained by PCR. The product containing restriction restriction site was linked to the prokaryotic expression vector PET-28b-TAT-V2 containing TAT transduction structure to obtain the recombinant expression vector TAT-c-Myc. The expression of TAT-c-Myc fusion protein was induced by IPTG. The expression product was identified by SDS-PAGE and purified by affinity chromatography. The specificity of the protein was detected by Western blot. The effect of fusion protein on human skin fibroblast was detected by immunofluorescence. Results the prokaryotic expression vector of TAT-c-Myc fusion protein was successfully constructed. After induction, the high expression of. Western was obtained. Blot analysis showed that the fusion protein had good specificity, and immunofluorescence assay showed that the fusion protein had the ability to be transferred into HSF cells quickly. Conclusion the results suggest that the fusion protein can be further induced into HSF cells by protein transduction. The cell provides the material basis.
【作者单位】：解放军第302医院国际肝病诊疗中心;解放军第302医院肝衰竭诊疗与研究中心;
【基金】：军队“十二五”重大课题(BWS11J075)
【分类号】：R329.2
【正文快照】： 诱导性多能干细胞(induced pluripotent stemcell,i PSC)是通过人工方法将某些特定的外源性转录因子导入体细胞或成体干细胞中,使体细胞或成体干细胞重新编程为具有无限增殖潜能和定向分化能力的、类似于胚胎干细胞的一类“人造干细胞”。2006年,Takahashi等[1]首次报道将oct4

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