【作者】 刘殿波；

【导师】 张七斤； 陈忠斌；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 本研究以人类新型冠状病毒NL63 (human coronavirus NL63, HCoV-NL63)木瓜样蛋白酶(papain-like protease, PLP)为研究对象,首先构建了木瓜样蛋白酶1 (papain-like protease 1, PLP1)的突变体D1225A和木瓜样蛋白酶2 (papain-like protease 2, PLP2)的突变体D1849A表达载体。通过Western Blotting技术证实,PLP1及其突变体C1062A、H1212A、D1225A对泛素(ubiquitin, Ub)分子不具有去泛素化(deubiquitinase, DUB)活性,PLP2却明显不同,野生型PLP2及突变体D1849A具有较强的DUB活性,而突变体C1678A、H1836A则丧失了此活性,同时对泛素样分子——干扰素刺激基因15(interferon stimulate gene 15, ISG15)进行检测,得到了相同的结论,提示PLP2的第1678位半胱氨酸和第1836位组氨酸是其DUB活性所必需的位点,第1849位天冬氨酸属于非必需位点。HCoV-NL63 PLP对宿主抗病毒天然免疫反应调控机制研究证实,PLP1及突变体C1062A、H1212A、D1225A均不能抑制干扰素-β(interferon-β, IFN-β)表达;PLP2和突变体D1849A却能强烈抑制IFN-β表达,但突变体C1678A、H1836却丧失了此功能。这表明,PLP2的干扰素拮抗活性与PLP2的DUB活性具有一定相关性。同时,对整个通路中关键蛋白调控的研究发现,PLP2与维甲酸诱导基因I (retinoic acid inducible gene I, RIG-I)、ERIS(endoplasmic reticulum IFN stimulator)相互作用,并利用自身的DUB活性特异性切除RIG-I、ERIS分子的多聚泛素化修饰,阻断其天然免疫反应,逃逸宿主防御系统。更多还原

【Abstract】 This project researches on papain-like protease of human coronavirus NL63. We constructed the mutants PLP1 D1225A and PLP2 D1849A. Western Blotting assay showed that PLP2 and PLP2 D1849A not PLP1 and its mutants have deubiquitinating(DUB) activity. Meanwhile, ubiquitin-like molecule the interferon sitimulate gene (ISG) was detected, we proved that PLP2 has deISGylation activity. It was suggested that the DUB activity of PLP2 is not dependent on its protease activity perfectly C1678A and H1836A are necessary for its DUB activity, but not D1849A.The research on PLP regulating innate immune response revealed that PLP2 not PLP1 supresses interferon (IFN) production, and this activity doesn’t dependent on PLP2 C1678 and PLP2 H1836. The results indicated that the IFN antagonist activity of PLP2 is related with its DUB activity. Further research manifested that PLP2 supresses IFN expression pathway by deubiquitinating of the retinoic acid inducible gene I (RIG-I) and the endoplasmic reticulum IFN stimulator (ERIS).更多还原