Molecular mechanism of Receptor-Interacting Protein 3 (RIP3) Enhancing Type I Interferons Production

2009以来，随着受体相互作用蛋白3（RIP3）介导细胞坏死信号通路的发现，跟RIP3相关的功能研究逐渐成为细胞生物学领域的研究热点。我们最近的研究发现RIP3能够显著促进I型干扰素的分泌，特别是促进双链DNA（dsDNA）诱导的I型干扰素的分泌。然而，RIP3是否参与调控DNA病毒诱导的I型干扰素的产生，并且RIP3是如何促进I型干扰素的分泌的，目前还不清楚。本项目拟通过利用CRISPR-Cas9介导的基因敲除技术构建RIP3敲除的细胞系，探讨RIP3在dsDNA和DNA病毒诱导的I型干扰素产生中的作用。我们还将进一步利用免疫沉淀（IP）和质谱联用的方法寻找RIP3的靶向分子，确定RIP3促...Receptor-interacting protein 3 has been reported to be a critical molecular in necroptosis, a form of programmed necrosis, in 2009. From then on, RIP3-related research has became a hot spot in cell biology area. In our recent study, we found that RIP3 plays a critical role in promoting type I interferons (IFNs) production, especially in double-standed DNA (dsDNA) induced IFNs production. However, ...学位：理学博士院系专业：物理科学与技术学院_生物化学与分子生物学学号：201417000

Exonuclease Ⅲ-mediated DNA Molecular Cloning

DNA克隆技术,作为最基本的现代分子生物学实验技术之一,已经成为生物医学研究领域的重要研究手段。传统的分子克隆方法需要经过限制性内切酶酶切和DNA连接酶连接的步骤,是否存在合适的酶切位点和DNA连接酶的效率成为影响克隆的重要限制因素。本文描述了一种由外切核酸酶Ⅲ介导的,以3'-5'外切核酸酶活性和细菌细胞内DNA修复机制为理论基础的DNA分子克隆方法,称为不依赖连接酶的分子克隆(ligation-independent cloning,LIC);证明了该方法的高效性和可靠性,并进一步对酶的用量、反应温度、反应时间、片段载体比例和量等多个参数进行了优化,建立了一种快速、简便和高效的DNA克隆方法。As a basic technique of modern molecular biology,molecular cloning has become an important tool for biomedical research. The traditional method for molecular cloning usually includes two key processes: restriction digestion and ligation. The success of a traditional cloning depends on suitable restriction sites and high ligation efficiency to a great degree. In the present study,we have developed a DNA cloning method mediated by exonuclease Ⅲ. Because this method is based on the 3' to 5' exonuclease activity of exonuclease Ⅲ and the intrinsic DNA repair machinery of bacteria,and is independent of DNA ligase,we designated it as ligation-independent cloning( LIC). We optimized the reaction parameters including the quantity of enzyme for each reaction,reaction time,reaction temperature and the ratio of vector to fragment,and found that this method showed high efficiency and reliability under optimal condition. We have thus established a rapid,simple and efficient DNA cloning method.国家自然科学基金项目(No.81402285)资助~

RIP1/RIP3 Binding to HSV-1 ICP6 Initiates Necroptosis to Restrict Virus Propagation in Mice

文章简介细胞坏死是一种由受体相互作用蛋白3(RIP3)和1(RIP1)介导的程序性死亡方式。本研究发现在RIP3缺失的小鼠细胞中,Ⅰ型单纯疱疹病毒(HSV-1)的繁殖效率远远高于在野生型细胞中的繁殖效率。这一结果表明:在小鼠细胞中,RIP3对于抵抗HSV-1的感染起到了非常重要的作用。进一步的研究发现,在感染HSV-1的小鼠细胞中,RIP1/3能够和HSV-1编码的ICP6蛋白发生相互作用;这一相互作用激活了RIP3介导的坏死信号,进一步引起细胞坏国家自然科学基金委重点项目;; 科技部973项目的资