重组人IL- 18的高效表达、纯化及诱导KG—1细胞产生高水平IFN-γ

目的 研究IL－18在大肠杆菌中高效表达及纯化工艺，并诱导KG－1 细胞产生IFN－γ。 方法利用RT－nPCR从正常人胎肝组织中扩增获得人IL－18基因，克隆于pThiohisA载体，转化宿主菌BL－21，1mmol/LIPTG；诱导，表达产物以包涵体形式存在，经2％Triton X－100洗涤，在 8mol/L尿素下变性阴离子柱层析，透析复性后再经凝胶过滤纯化，通过纯化产物诱导入骨髓瘤单核细胞 KG－l（ATCC CCL－246）产生 IFN－γ，并检测其生物学活性。结果 获得了在大肠杆菌中的稳定高效表达，表达的目的蛋白占菌体总蛋白的25％左右，纯化后纯度可达95％以上，具有显著的刺激细胞产生IFN－γ的活性。结论 制备具有生物学活性高纯度的rhIL－18方法的建立，为基础研究与临床应用开发奠定了基础。

High Expression and Purification of Recombinant Human IL-18 and Bioassay of IFN-γ Secreted with KG-1 Cells Under Induction of Expressed Product

Abstract: Objective To highly express rhIL - 18 in E. coli and establish the methods for purification and bioassay. Methods hIL-18 cDNA was amplified from normal human embryo liver tissue by RT-PCR, cloned into pThiohis A vector, transformed to E. coli BL-21, and expressed under the induction of 1mol/L IPTG. The expressed product existing in the form of inclusion body was washed with 2% Triton-X-100, dissolved in 8mol/L urea and purified by denatured anion exchange chromatography. After being renaturalized by dialysis, the expressed product was further purified by Superdex-75 size exclusion chromatography. The biological activity of purified product was evaluated by detecting the INF-γlevel secreted with human myelomonocyte KG-1 under the induction of it.Results rhIL-18 was stably and highly expressed in E. coli. The expressed product contained about 25% of total somatic protein and reached a purity of above 95% after being purified. It induced the secretion of IFN-γsignificantly. Conclusion A method for preparing rhIL-18 with biological activity and high purity is established and can be scaled up. The method laid a foundation of the basic research and clinical application of rhIL-18.