融合蛋白TOP_3原核表达载体的构建、表达及纯化研究

Construction,expression and purification of fusion protein TOP_3 in prokaryotic expression system

目的:利用HIV病毒TAT蛋白转导结构域、人缺氧诱导因子1α氧依赖降解结构域以及CASPASE3构建融合蛋白的原核表达载体,并在大肠杆菌中表达及纯化。方法:分别构建原核表达质粒pET-28a(+)-TAT、pET-28a(+)-TAT-ODD、pET-28a(+)-TAT-ODD-CASPASE3(pET-28a(+)-TOP3);在大肠杆菌BL21(DE3)pLysS内,利用镍-亚硝胺乙酸-组氨酸(Ni-NTA-His)亲和层析法对重组蛋白TOP3进行纯化。结果:成功构建了pET-28a(+)-TOP3的原核表达载体,经过优化表达和纯化条件,融合蛋白在IPTG诱导下获得特异性表达,纯化得到了高纯度的目的蛋白。结论:融合蛋白TOP3的成功表达及纯化,为该融合蛋白应用于肿瘤的治疗研究奠定了理论基础。

Objective：To construct a prokaryotic expression vector for a fusion protein, TAT protein transduetion domain （PTD）.the ODD domain of hypoxia inducible factor1α（HIF-1α） and cysteinyl aspartatespecific protease 3, and then express and purify the fusion protein. Methods ：The expression plasmids pET-28a （＋）-TAT, pET-28a （ ＋ ）-TAT-ODD, pET-28a （ ＋ ）-TAT-ODD-CASPASEs （pET-28a （ ＋ ）-TOP3 ） were constructed respectively, and pET-28a （＋）-TOP3 was transformed into E coll. BL21 （DE3）pLysS strain to be induced by IPTG. The obtained proteins were analyzed by SDS-PAGE. The fusion protein was purified with Ni- NTA-His affinity chromatography. Results： The recombinant plasmids were constructed successfully. The objective fusion protein was obtained by optimizing the conditions for expression and purification. Conclusion： The successful expression and purification of the fusion protein TOP3 has laid the foundation for using it to studing tumor therapy.