小鼠DNA依赖的干扰素调节因子激活物分子结构特征及其功能

Molecular structural characteristics and the functions of mouse DNA-dependent activator of interferon-regulatory factors

目的研究小鼠DNA依赖的干扰素调节因子激活物(DAI)的分子结构特征以及抗病毒天然免疫应答与信号转导的分子机制。方法应用反转录PCR技术从小鼠脾脏淋巴细胞中克隆DAI基因,并利用生物信息学软件对其遗传演化关系及分子结构特征进行分析;双荧光素酶报告系统检测小鼠DAI受到聚脱氧腺苷酸-脱氧胸苷酸[poly(d A-d T)]和聚脱氧鸟苷酸-脱氧胞苷酸[poly(d G-d C)]作用后β干扰素(IFN-β)启动子和核因子κB(NF-κB)调控元件启动的荧光素酶活性。结果克隆的小鼠DAI基因开放读框全长为1236 bp,编码411个氨基酸,与牛、猪、大鼠等哺乳动物DAI相应序列的同源性为60%、63.1%、84%,由2个Z-DNA结合结构域Zα和Zβ、DNA结合区3(D3)和信号转导结构域(SD)组成。小鼠DAI受到poly(d A-d T)作用后,IFN-β启动子和NF-κB调控元件起始的相对荧光素酶活性均显著增加;而小鼠DAI受到poly(d G-d C)作用后,只有NF-κB起始的相对荧光素酶活性显著增加,IFN-β启动子起始的相对荧光素酶活性并无显著变化。结论小鼠DAI能识别富含A/T或C/G的DNA序列,可能在宿主抗DNA病毒感染的天然免疫反应中发挥重要作用。

Objective To study the structural characteristics of the mouse DNA-dependent activator of interferon-regulatory factors（ DAI） and its related molecular mechanism in anti-viral innate immune responses and signal transduction. Methods The coding sequence of mouse DAI gene was amplified from splenic mononuclear cells by reverse transcription-PCR,and the genetic evolution and molecular structure of the mouse DAI gene were analyzed by bioinformatics softwares. After mouse DAI was stimulated by poly（ d A-d T） and poly（ d G-d C）,the nuclear factor κB（ NF-κB） and interferon-beta（ IFN-β） promoterdriven luciferase activity were detected by dual-luciferase reporter assay system. Results The open reading frame（ ORF） of the cloned mouse DAI sequence was 1236 bp,encoding 411 amino acids,which exhibited identity with the corresponding sequences of cattle,pig,rat and other mammals ranging from 60% 、63. 1% 、84%,and it contained two Z-DNA domains（ Zαand Zβ）,DNA binding region（ D3） and signaling domain（ SD）. The stimulation of poly（ d A-d T） increased the expressions of mouse DAI activated transcription factors NF-κB and IFN-β promoter. However,the stimulation of poly（ d G-d C） only induced the activation of NF-κB but not IFN-β promoter. Conclusion Mouse DAI as an important cytosolic DNA sensor,is responsible for the recognition of A / T or G / C-rich DNA derived from DNA virus. It may play an important role in anti-viral innate immune responses.