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目的构建小鼠白细胞介素18(interleukin18,IL-18)的真核表达质粒,并检测其表达的IL-18的生物学活性。方法采用RT-PCR获得小鼠IL-18基因,通过EcoRⅠ和HindⅢ双酶切及连接反应,构建pEGFP-mIL-18真核表达载体,重组载体经过限制性内切酶、PCR及DNA序列测定等证实连接片段的正确性后,转染HEK293细胞系,荧光显微镜观察GFP-mIL-18融合蛋白的表达,收集转染后72h的上清液,MTT法检测上清液中IL-18表达产物的生物学活性。结果酶切分析、PCR鉴定、DNA测序表明成功地构建了pEGFP-mIL-18真核表达载体,MTT法证明表达产物有刺激小鼠脾细胞增殖的功能。结论所获pEGFP-mIL-18真核表达载体能在体外表达具有生物学活性的IL-18,为进一步研究IL-18的功能和运用奠定了基础。
Objective To construct the eukaryotic expression plasmid of mouse interleukin 18 (IL-18) and test the biological activity of its expressed IL-18. Methods The mouse IL-18 gene was obtained by RT-PCR. The eukaryotic expression vector pEGFP-mIL-18 was constructed by restriction endonuclease digestion and ligation with EcoRⅠand HindⅢ. The recombinant vector was confirmed by restriction enzyme, PCR and DNA sequencing After confirming the correctness of the ligated fragment, HEK293 cells were transfected, and the expression of GFP-mIL-18 fusion protein was observed under a fluorescence microscope. The supernatant was collected 72h after transfection. The expression of IL-18 in supernatant was detected by MTT assay Biological activity. Results Enzyme digestion analysis, PCR identification and DNA sequencing showed that the eukaryotic expression vector pEGFP-mIL-18 was successfully constructed. The expressed product proved to be able to stimulate the proliferation of mouse spleen cells. Conclusion The eukaryotic expression vector pEGFP-mIL-18 can express biologically active IL-18 in vitro, which lays the foundation for further study on the function and utilization of IL-18.