带有穿膜结构域的转录因子蛋白Oct4和Sox2的融合表达

目的:克隆、表达及纯化带有穿膜结构域的转录因子蛋白Oct4和Sox2。方法:根据GenBank中的Oct4和Sox2基因序列,在其3’端引入穿膜结构域11R,并在其两端引入NdeⅠ和XhoⅠ酶切位点,进行全基因合成;将目的基因克隆至pET41a载体,进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21(DE3),经IPTG诱导表达后,对表达产物进行Western印迹鉴定;最后用Ni-NTA亲和层析柱对所获目的蛋白进行纯化。结果:质粒酶切鉴定结果表明带有目的基因的重组质粒构建成功;SDS-PAGE结果显示有相对分子质量约42×103和38×103的特异性蛋白表达条带,经Western印迹证实为目的蛋白;用Ni-NTA亲和层析柱纯化后,得到均一的Oct4和Sox2目的蛋白。结论:得到带有穿膜结构域的转录因子融合蛋白Oct4和Sox2,为今后安全开展诱导性多能干细胞研究奠定了基础。

Fusion Expession and Purification of Recombinant Oct4 and Sox2 with Cell-Penetrating Peptide

Objective: To express and purify of recombinant fusion proteins Oct4 and Sox2 with cell-penetrating peptide.Methods: A poly-arginine(11R) was designed and fused to the C terminus of Oct4 and Sox2,which were cloned into expression vector pET41a.After sequence analysis,the acquired recombinant plasmids were trans formed into E.coli BL21(DE3) and target proteins were expressed by inducing with IPTG.Then,the proteins were identified by Western blotting and purified with Ni-NTA purification system.Results: The recombinant plasmids were successfully constructed.Western blot analysis showed that the fusion proteins were expressed.SDS-PAGE showed that the expressed fusion proteins were successfully purified from the lysate of E.coli.Conclusion: The re combinant proteins Oct4 and Sox2 with cell-penetrating peptide were successfully obtained,which will lay the foun dation for safely inducing of induced pluripotent stem cells in vitro.