水牛iPS细胞转录因子Sox2及协助蛋白HA2-TAT的原核表达

Prokaryotic expression of buffalo iPSCs transcription factor Sox2 and assist protein HA2-TAT

为探讨干细胞转录因子与穿膜肽融合表达蛋白对水牛体细胞诱导重编程的可行性,本试验对水牛iPS细胞转录因子Sox2与细胞穿膜肽HIV TAT进行融合表达,以获得具有自主穿膜功能的Sox2蛋白,建立非转基因水牛iPS生产技术体系。首先人工合成了HA2-TAT序列,并将pET-32a(+)质粒改造为pET-HA2-TAT基础表达载体,再通过双酶切定向克隆将水牛Sox2基因插入pET-HA2-TAT得到原核表达载体pET-NLS-Sox2-TAT;重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导,用SDS-PAGE电泳和Western blot检测融合蛋白表达,再用Ni 2+柱进行纯化融合蛋白。结果表明,成功表达了融合蛋白HA2-TAT(24 400)和NLS-Sox2-TAT(57 700);纯化蛋白NLS-Sox2-TAT在咪唑浓度为365.30mmol/L时,出现蛋白洗脱峰,经Western blot验证表达的融合蛋白具有免疫原性。

To obtain Sox2 protein which could penetrate cell membrane,buffalo iPSCs（induced pluripotent stem cells） transcription factor Sox2 and cell-penetrating peptide HIV TAT were fusingly expressed.First,HA2-TAT fragment was synthesised to construct a basic expressing plasmid,pET-HA2-TAT.Then buffalo Sox2 gene was subcloned to pET-HA2-TAT,forming a recombinant plasmid pET-NLS-Sox2-TAT.It was transformed into E.coli BL21（DE3） and the fusion protein was expressed with induction of IPTG.SDS-PAGE analysis and Western blot was performed to detect the fusion protein which was isolated and purified by Ni2＋ protein purificating column.The results showed that the fusion protein HA2-TAT（24 400）and NLS-Sox2-TAT（57 700） could high efficiently expressed.When the imidazole at the density of 365.30 mmol/L,fusion protein NLS-Sox2-TAT emerges eluting peak.The fusion protein could be detected with biology antigenicity by Western blot.