【摘要】 目的:研究表面活性剂十六烷基三甲基溴化铵(CTAB)和β-环糊精(β-CD)组成的人工分子伴侣系统辅助鸡IL-18重组蛋白的复性,以提高鸡IL-18重组蛋白复性率,获得更多具有良好活性的蛋白。方法:将重组原核表达质粒pGEX-mChIL-18转化宿主细胞大肠杆菌BL21(DE3),并用IPTG于37℃诱导培养获得表达。表达产物主要以包涵体的形式存在。包涵体经超声波破碎、洗涤后以6mol/L的盐酸胍溶解,使蛋白彻底变性,然后利用人工分子伴侣系统辅助蛋白复性。复性产物经透析纯化后,利用淋巴细胞增殖试验来检测其活性。结果:经SDS-PAGE分析表明,表达产物是与ChIL-18重组蛋白相符的Mr约44000的蛋白条带。利用人工分子伴侣系统辅助复性,鸡IL-18重组蛋白获得了42·54%复性率。鸡淋巴细胞增殖试验表明,表达产物对鸡淋巴细胞具有明显诱导增殖作用。结论:人工分子伴侣系统能够较好地辅助鸡IL-18重组蛋白复性,获得较高的复性率。其产物具有良好的生物学活性,为下一步鸡IL-18重组蛋白的应用研究奠定了试验基础。更多还原

【Abstract】 AIM: To study the technique of boosting the renaturation yield of rChIL-18 by using aritificial molecular chaperone composed of cetyl trimethyl ammonium bromide(CTAB) and β-cyclodextrin(β-CD). METHODS: The recombinant plasmid of mChIL-18 prokaryotic expression was transformed into E.coli BL21(DE3) strain and then induced by IPTG at 37℃. The recombinant mChIL-18 was expressed efficiently in inclusion bodies in E.coli. After crushed and washed, the inclusion bodies were thoroughly denatured with 6 mol/L of guanidine hydrochloride, and then the artificial molecular chaperone was used to promote protein refolding. After the rehabilitation of products was purified by bag filter, its activity was detected by lymphocyte proliferation assays. RESULTS: The SDS-PAGE analysis indicated the expressed ChIL-18 protein had molecular weight of 44 000. The expressed product existed in the form of inclusion body.Two protein bands of Mr 44 000 and 26 000 appeared on SDS-PAGE gel. The percentage of renaturation was 42.54 with artificial molecular chaperone.The results of MTT assay showed the expression of ChIL-18 protein in E.coli BL21(DE3) greatly induced the proliferation of chicken T lymphocytes. CONCLUSION: The artificial chaperone technique can obviously boost the renaturation yield of rChIL-18. The purified and expressed product of fusion chicken Interleukin-18 gene in E.coli have relativity high bioactivity.更多还原