土拨鼠白细胞介素15的克隆、表达和活性分析

目的对土拨鼠白细胞介素15（woodchuck interleukin 15，wIL-15）分子进行克隆、表达并鉴定其活性，为进一步研究IL-15在嗜肝病毒感染中的作用以及评价IL-15用于治疗慢性HBV感染奠定基础。方法用IL-15的保守引物将wIL-15基因克隆、测序并进行分析，构建wIL-15的原核表达质粒，在大肠杆菌中表达，分离纯化后用淋巴细胞增殖试验检测其生物学活性。结果wIL-15的完整编码序列为489nt（与其他哺乳动物IL-15编码序列的同源性高达78％～87％），编码162AA长度的前体蛋白（与其他哺乳动物IL-15蛋白的同源性高达70％～80％），其信号肽为N端的48AA，成熟蛋白由114AA组成。土拨鼠IL-15与灵长动物IL-15的同源性高于啮齿动物。带有His-tag的成熟蛋白，可以在大肠杆菌中表达，分离纯化后，可以刺激Con A活化的小鼠脾细胞和土拨鼠PBMC增殖（其SI值可高达10以上），并存在明显的剂量依赖性。结论（1）wIL-15与其他哺乳动物IL-15高度同源，并且与灵长类动物的IL-15更接近。（2）wIL-15可以在大肠杆菌中表达，并在纯化后能刺激小鼠和土拨鼠淋巴细胞增殖，表现出较好的生物学活性。

Cloning, expression and biological activity analysis of woodchuck interleukin 15

Objective Cloning, sequence analysis and molecular characterization of woodchuck interleu-kin 15(wIL-15), for further study of its roles in hepadnavirus infection and the potential application in treatment of chronic HBV infection. Methods Cloning, sequencing of wIL-15 molecules, using IL-15 conserved primers, developing wIL-15 expressing plasmids, expressing of wIL-15 in E. coli, determination the biological activity of expressed wIL-15 after purification. Results The complete coding sequence of wIL-15 is 489 nt; the predicted precursor is 162AA, with a 48AA long signal peptide. His-tagged mature protein is expressed in E.coli, and it can stimulate the proliferation of mice and woodchuck lymphocytes in a dose-dependent manner after purification. Conclusions (1) The complete coding sequence of wIL-15 is highly homology to its counterpart of other mammalian species. (2) wIL-15 can be expressed in E. coli, and can stimulate the proliferation of lymphocytes after purification, so the expressed protein remains its biological activity.