土拨鼠白细胞介素15的克隆、表达和活性分析

Cloning, expression and biological activity analysis of woodchuck interleukin 15

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489 nt(与其他哺乳动物IL-15编码序列的同源性高达78％-87％),编码162 AA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48 AA,成熟蛋白由114 AA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。

Objective Cloning, sequence analysis and molecular characterization of woodchuck interleukin 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.