猪RNA识别受体DHX15的克隆及其在诱导Ⅰ型IFNs中的作用研究

发布时间：2019-05-13 22:04

【摘要】：DHX15是近几年发现的胞浆RNA识别受体,属于DEXD/H-解旋酶家族,能够通过C端的解旋酶结构域与polyI:C或病毒的dsRNA结合,然后通过N端的DEXDc结构域与VISA相互作用,激活IRF3、NF-κB和MAPK,诱导IFN-β、IL-6和TNF-a的分泌。迄今为止,仅人和小鼠的DHX15被鉴定,猪DHX15(porcineDHX15,pDHX15)的研究尚未报道。因此,本研究首先克隆pDHX15基因,然后对其遗传进化关系和分子结构特征进行分析,继而对其在诱导I型干扰素中的作用进行了初步研究。本研究主要内容如下:1.pDHX15基因的克隆与序列分析应用RT-PCR技术从猪外周血淋巴细胞(PBMCs)中克隆了pDHX15基因,然后将其插入pJET1.2/blunt克隆载体中,构建了pJET1.2-pDHX15。序列分析发现,pDHX15基因开放阅读框架(ORF)全长2388bp,编码795个氨基酸,与人类、小鼠、大鼠、牛、羊、犬、猫、马等哺乳动物的同源性都在99.4%以上,而与两栖类非洲爪蟾、中华鳖、鸟类绿头鸭、鱼类斑马鱼的同源性相对较低,分别为92.2%、95.3%、94.2%和91.3%;结构分析显示,pDHX15含有N端的两个松散结构域、DEAH-box保守结构域、HELICc解旋酶活性结构域、HA2结构域以及DUF结构域。2.pDHX15的组织差异表达与亚细胞定位分析应用半定量RT-PCR和Real-timePCR技术分析pDHX15的mRNA在不同组织的表达情况。结果显示,pDHX15mRNA广泛表达于所有检测的组织中,包括外周血、心脏、肝脏、脾脏、肺脏、肾脏、小肠、淋巴结和皮肤,其中在肺脏、淋巴结和小肠中表达量最高,心脏、脾脏、肾脏和皮肤次之,外周血和肝脏的表达量最少。此外,为了研究pDHX15的亚细胞定位,将构建的与EGFP标签融合表达的pDHX15真核表达载体pEGFP-pDHX15分别与靶向定位于细胞核、内质网、高尔基体和线粒体的红色荧光质粒共转染HEK293T细胞,24h后用激光共聚焦显微镜确定pDHX15的亚细胞定位。结果显示,在转染pEGFP-pDHX15的HEK293T细胞内,带有绿色荧光的pDHX15弥散性的分布于整个细胞内,能够分别与细胞核、内质网、线粒体、高尔基体靶向定位的红色荧光重叠。3.pDHX15在诱导I型干扰素产生中的作用为了研究pDHX15在诱导I型干扰素产生中的作用,将构建的真核表达载体pCAGGS-HA-pDHX15、pCAGGS-pDHX15-△DUF、pCAGGS-pDHX15-△DUF-△HA2以及pCAGGS-pDHX15-DEXDc分别与猪源IFN-β启动子以及转录调控元件IRF3和NF-κB荧光素酶报告质粒共转染HEK293T细胞。结果显示,在poly(I:C)刺激下,pCAGGS-HA-pDHX15以剂量依赖的方式显著活化转录因子IRF3、NF-κB以及IFN-β的产生;当用siRNA敲降pDHX15后,能够显著降低poly(I:C)诱导IRF3和NF-κB以及IFN-β的活化。此外,pDHX15结构域缺失突变显示,DUF结构域和HELIC酶活性结构域属于负向调控结构域,能够负调节poly(I:C)诱导IRF3和NF-κB以及IFN-β的激活。但是,HA2结构域和DEXD结构域是正向调控结构域,能够正调节poly(I:C)诱导IRF3、NF-κB以及IFN-β的产生。进一步研究发现,pDHX15以剂量依赖的方式通过招募关键接头分子VISA和MITA激活IRF3和NF-κB,进而诱导IFN-β的表达。综上,pDHX15是猪的一个天然免疫RNA识别受体,在诱导I型IFNs的产生中具有重要作用。
[Abstract]:DHX15 is a cytoplasmic RNA recognition receptor found in recent years, and belongs to the family of DEXD/ H-helicase. The DHX15 can be combined with the dsRNA of polyI: C or the virus through the helicase domain at the C end, then interacts with the VISA through the DEXDc domain of the N end, activates the IRF3, NF-EMAB and MAPK, and induces the IFN-1, The secretion of IL-6 and TNF-a. To date, only human and mouse DHX15 has been identified, and the study of porcine DHX15 (pDHX15, pDHX15) has not been reported. Therefore, the pDHX15 gene was cloned first, then the genetic evolution and molecular structure of the pDHX15 gene were analyzed, and then the role of the pDHX15 gene in the induction of type I interferon was studied. The main contents of this study are as follows:1. Cloning and sequence analysis of 1. pDHX15 gene. The pDHX15 gene was cloned from peripheral blood lymphocytes (PBMCs) by RT-PCR, and then inserted into pJET1.2/ blunted cloning vector to construct pJET1.2-pDHX15. The sequence analysis showed that the full length of the open reading frame (ORF) of the pDHX15 gene was 2388 bp, encoding 795 amino acids, and the homology with the mammals such as human, mouse, rat, cattle, sheep, dog, cat, horse and other mammals was over 99.4%, and compared with the amphibian African claw, the Chinese soft-shelled turtle and the bird's green-headed duck. The homology of the fish zebrafish is relatively low, 92.2%, 95.3%, 94.2% and 91.3%, respectively; the structural analysis shows that the pDHX15 contains two loose domains at the N end, the DEAH-box conserved domain and the HELICc helicase active domain, The expression of pDHX15 mRNA in different tissues was analyzed by semi-quantitative RT-PCR and Real-time PCR. The results show that the pDHX15mRNA is widely expressed in all the detected tissues, including peripheral blood, heart, liver, spleen, lung, kidney, small intestine, lymph node and skin, wherein the expression of pDHX15mRNA is the highest in the lung, lymph node and small intestine, and the heart, spleen, kidney and skin are the second. The expression of peripheral blood and liver is the least. In addition, in order to study the subcellular localization of pDHX15, the pDHX15 eukaryotic expression vector pEGFP-pDHX15, which is constructed by fusion expression with the EGFP label, is respectively and targeted to be located in the nucleus and the endoplasmic reticulum, HEK293T cells were co-transfected with the red fluorescent plasmids of the Golgi and the mitochondria, and the subcellular localization of pDHX15 was determined by a laser confocal microscope at 24 h. The results show that, in the HEK293T cells transfected with pEGFP-pDHX15, the pDHX15 with green fluorescence is distributed in the whole cell, and can be respectively connected with the nucleus, the endoplasmic reticulum and the mitochondria. the role of pDHX15 in inducing the production of type I interferon is to study the role of pDHX15 in inducing the production of type I interferon, and the constructed eukaryotic expression vector pCAGGS-HA-pDHX15, pCAGGS-pDHX15-pDHDUF, The pCAGGS-pDHX15-DDUF-pDHA2 and pCAGGS-pDHX15-DEXDc were co-transfected with the porcine source IFN-yeast promoter and the transcription regulatory element IRF3 and the NF-VIB luciferase reporter plasmid, respectively, to HEK293T cells. The results showed that, under the stimulation of poly (I: C), pCAGGS-HA-pDHX15 significantly activated transcription factors IRF3, NF-B and IFN-B in a dose-dependent manner; after pDHX15 was knocked down with siRNA, the activation of poly (I: C)-induced IRF3 and NF-EMAB and IFN-1 was significantly reduced. In addition, that deletion mutation of the pDHX15 domain show that the DUF domain and the HELIC enzyme activity domain belong to the negative regulatory domain, and the activation of the poly (I: C)-induced IRF3 and NF-EMAB and the IFN-antigen can be negatively regulated. However, the HA2 domain and the DEXD domain are forward regulatory domains that are capable of modulating poly (I: C) to induce the production of IRF3, NF-B, and IFN-C. Further studies have found that pDHX15 induces the expression of IFN-antigen in a dose-dependent manner by the recruitment of key linker molecules VISA and MITA to activate IRF3 and NF-B. In general, pDHX15 is a natural immune RNA recognition receptor for pigs and plays an important role in inducing the production of type I IFNs.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828;Q78

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