重组Prohibitin融合蛋白包涵体的纯化

Purification of Recombinant Prohibitin Fusion Protein from Inclusion Body

[目的]获得高纯度具有免疫活性的Prohibitin融合蛋白。[方法]异丙基-β-D硫代半乳糖苷(IPTG)诱导重组大肠杆菌BL21(pET30(a+)-prohibitin)表达,其融合蛋白包涵体经过洗涤、变性、复性后,用Ni-NTAAgarose亲和层析柱分离纯化。通过12%SDS-PAGE胶和Bradford法检测其纯度和目的蛋白含量,用ELISA对纯化后的蛋白进行鉴定。[结果]从融合蛋白包涵体中纯化到具有免疫活性的目的蛋白,其纯度达90%以上,含量约0.3mg/ml。[结论]建立了有效纯化融合蛋白包涵体的方法,为对Prohibitin进一步的结构和功能研究奠定基础。

[Purpose]To obtain highly purified Prohibitin fusion protein with immunocompetenee. [Methods] After inducing E.Coli BL21 （pET30 （＋ ）-prokibitin ）by isopropylthio-β-D-galactoside （IPTG）, inclusion body was washed, denatured and renatured, then the recombinant fusion protein was purified by Ni-NTA Agarose affinity column. The purity and concentration were measured by 12% SDS-PAGE and Bradford method, then verified protein with ELISA. [Results] Prohibitin fusion protein with immunocnmpetence was obtained from inclusion body. Purity was higher than 90% and concentration was about 0.3mg/ml. [Conclusions ]An effective method of fusion protein purification from the inclusion bodies is developed, which is the basis for further study on its structure and function.