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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。
OBJECTIVE: To study the refolding of chicken IL-18 recombinant protein by artificial chaperone system composed of surfactant cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) IL-18 recombinant protein renaturation rate, get more good activity of the protein. Methods: The recombinant plasmid pGEX-mChIL-18 was transformed into E. coli BL21 (DE3) and induced by IPTG at 37 ℃. The expression product mainly exists in the form of inclusion body. Inclusion body by ultrasonic broken, washed with 6mol / L guanidine hydrochloride to dissolve, the protein denaturation, and then use the artificial molecular chaperone system to help protein renaturation. Refolded products were purified by dialysis, the use of lymphocyte proliferation test to test its activity. Results: SDS-PAGE analysis showed that the expression product was about 44000 protein bands corresponding to ChIL-18 recombinant protein. Artificial molecular chaperone system assisted renaturation, chicken IL-18 recombinant protein obtained 42.54% refolding rate. Chicken lymphocyte proliferation test showed that the expression product of chicken lymphocytes significantly induced proliferation. Conclusion: The artificial chaperone system can better complement the chicken IL-18 recombinant protein and obtain a higher refolding rate. The product has good biological activity, laying a foundation for the further study on the application of chicken IL-18 recombinant protein.