人白介素-7重组蛋白的原核表达、纯化及活性测定

Prokaryotic expression,purification and activity of human interleukin-7 recombinant protein

目的:构建重组质粒p ET32a-h IL-7,诱导表达,纯化并鉴定目的蛋白。方法:将人白介素-7基因克隆到原核表达载体p ET32a(+)上,得到重组质粒p ET32a-h IL-7,转化大肠杆菌BL21(DE3),经IPTG诱导得到融合蛋白Trx-h IL-7。融合蛋白主要以包涵体形式存在。包涵体通过复性、酶切、纯化等步骤处理。利用Western blot鉴定重组蛋白,对其生物学活性用其依赖性细胞株2E8增殖实验测定。结果:成功构建重组质粒p ET32a-h IL-7。包涵体通过透析复性,肠激酶切,镍柱纯化等步骤得到重组蛋白人IL-7。纯化的重组蛋白经Western blot鉴定能与抗人的IL-7抗体特异性结合。经体外细胞实验检测证明,纯化的重组蛋白具有生物学活性。结论:得到了有活性的重组蛋白IL-7,为研究其功能奠定了基础。

Objective： To acquire recombinant human interleukin 7 prokaryotic expressing plasmid,fusion protein is expressed,purified and identified. Methods： The h IL-7 gene was inserted into the prokaryotic expressing vector p ET-32a（ ＋） to acquire p ET32-h IL-7 and transformed into BL21（ DE3） cells. IPTG induced the expression of fusion protein Trx-IL-7. The induced product was washed,dissolved and purified by affinity chromatogarphy under renaturing condition. IL-7 was identified by Western blot,and the biologic activity was detected by proliferation of IL-7 dependence cell 2E8. Results： The recombinant plasmid p ET32 / rhl L-7 has been constructed correctly. The recombinant hl L-7 was gained after gradient dialysis,enzyme digestion and purified,which has the right immunology specificity and biologic activity. Conclusion： The recombinant human interleukin-7 with biologic activity has been acquired,which lay the foundation for the further study of function of IL-7.