【作者】 刘琴；

【导师】 方六荣；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 DDX3X是含DEAD-box家族的RNA解旋酶成员之一。DEAD-box解旋酶参与许多的生物学过程,如：调节转录、RNA剪切、核运输、控制mRNA的翻译等。研究表明DDX3X参与天然免疫应答,增强抗病毒物质的产生；另一方面,DDX3X有可能是病毒达到天然免疫逃避的目的的手段之一。PRRSV是一种单股正链RNA病毒,属于动脉炎病毒科属,引起PRRS,给世界养猪业造成了极大的损失。生产有效的PRRSV疫苗是养猪业迫切的需求。目前还不清楚猪DDX3X的功能及其对PRRSV增殖的影响,鉴于此,本研究主要是分析猪DDX3X的功能及其对PRRSV增殖的影响,主要研究内容如下：1.猪DDX3X基因的扩增、序列分析、及进化树的绘制通过对猪基因EST数据库的分析,设计特异性引物,PK-15细胞RNA为模板,RT-PCR扩增获得猪DDX3X基因序列。分析发现猪DDX3X的核苷酸序列全长为1986bp,编码662个氨基酸,与鼠、人、牛、鸡、日本血吸虫的氨基酸序列同源性分别为97%、97%、98%、90%、53%,系统进化树分析虽然表明不同物种DDX3X的氨基酸序列存在一定差异,但是DDX3X在进化上还是比较保守的,并且猪DDX3X与牛DDX3X的亲缘关系最近。2.猪DDX3X的亚细胞定位将猪DDX3X基因插入真核表达载体pEGFP-C2中构建猪DDX3X与EGFP融合的真核表达质粒pEGFP-C2-DDX3X,转染PK-15细胞后发现猪DDX3X主要定位于胞浆,没有发现猪DDX3X在细胞核中的定位。3.猪DDX3X参与IFN-p诱导以猪DDX3X的真核表达质粒pCAGGS-HA-DDX3X转染PK-15细胞,通过荧光定量检测证实猪DDX3X能上调IFN-βmRNA水平的表达。进一步利用猪DDX3X的干扰分子sipDDX3X干扰细胞内源性猪DDX3X的表达,检测猪DDX3X对SeV/poly(dA:dT)诱导IFN-β的影响,结果猪DDX3X的敲除明显下调SeV/poly(dA:dT)诱导的IFN-βmRNA水平。利用双荧光素酶检测系统证实猪DDX3X能显著激活IFN-p启动子。4.猪DDX3X以剂量依赖的形式促进TBK1/IPS-1激活IFN-p启动子利用红荧光蛋白和绿荧光蛋白作标记证实猪DDX3X与TBK1/IPS-1在PK-15细胞中均存在共定位现象,由此说明猪DDX3X与TBK1/IPS-1存在相互作用。进一步利用双荧光素酶检测系统证实猪DDX3X促进TBK1/IPS-1激活IFN-β启动子,并呈剂量依赖的形式,也进一步证实了猪DDX3X与TBK1/IPS-1存在相互作用。5.猪DDX3X与PRRSV的相互作用将pEGFP-C2-DDX3X真核表达质粒转染Marc-145细胞,然后接种PRRSV,DAPI染色后用激光共聚焦显微镜观察证实PRRSV感染不改变猪DDX3X在Marc-145细胞中的定位。在SJPL细胞中瞬时表达猪DDX3X,用绝对定量和TCID50检测发现猪DDX3X抑制PRRSV的增殖,进一步实验发现PRRSV能够上调PAM细胞中猪DDX3X的表达。更多还原

【Abstract】 DDX3X is a RNA helicase, which belongs to the DEAD-box family of RNA helicases. DEAD-box helicases participate in many cellular processes, such as translational and transcriptional regulation, RNA splicing and nucleus export. On one hand, the results show that DDX3X takes part in the natural immune response and enhances the production of antiviral substances. On the other hand, DDX3X may be one of the means that virus achieve the purpose of the natural immune evasion.PRRSV is a single-strand RNA virus and is a member of the Arteritis virus. PRRSV causes PRRS, which brings great challenges to the pig industry around the world. Production of an effective vaccine is urgently required in the pig industry. Currently, we don’t understand the functions of porcine DDX3X and the effect on the proliferation of PRRSV. So this study aimed to analyze the function of porcine DDX3X and the effect on the proliferation of PRRSV. The main contents were as follows:1. The Cloning, sequence analysis and phylogenetic tree construction of the porcine DDX3X geneThe porcine EST database was analyzed, and then the specific primers were designed. RNA of PK-15 cells was used as the template and the porcine DDX3X gene sequence was amplified via RT-PCR. The result of sequencing analysis indicated that the full length of porcine DDX3X’s nucleotide was 1986bp and the open reading frame had 662 amino acids. And it had a amino acid homology of 97%,97%,98%,90%, and 53% with rodents, human, cattle, chicken and schistosoma japonicum, respectively. Although the result of phylogenetic tree analysis showed that there were some differences among the amino acid sequences of different species DDX3X, DDX3X was quite conserved in evolution, and porcine DDX3X had the closest relationship with cattle DDX3X.2. The subcellular localization of porcine DDX3XThe porcine DDX3X gene was inserted into the eukaryotic expression vector PEGFP-C2 to construct the eukaryotic expression plasmid of pEGFP-C2-DDX3X, and it was transfected into the PK-15 cells. Then we found that porcine DDX3X was mainly located in the cytoplasm, not in the nucleus.3. porcine DDX3X participated in the IFN-βproductionpCAGGS-HA-DDX3X was transfected into the PK-15 cells, then it was confirmed that porcine DDX3X could increase the levels of IFN-βmRNA expression by relative quantitative detection. Further, the siRNA named sipDDX3X which interfered with the endogenous porcine DDX3X was designed and was transfected into the PK-15 cells. Then the porcine DDX3X’s effect on the IFN-βwhich was induced by SeV/poly(dA:dT) was detected. The result showed that the IFN-βmRNA levels induced by SeV/poly(dA:dT) were significantly decreased after the endogenous porcine DDX3X was knocked out. Meanwhile porcine DDX3X activated the IFN-βpromoter via dual luciferase assay system.4. The porcine DDX3X promoted TBK1/IPS-1 to activate the IFN-βpromoter in a dose-dependent mannerporcine DDX3X and TBK1/IPS-1 which were marked by the red fluorescent protein and green fluorescent protein co-localized in PK-15 cells. The result indicated that porcine DDX3X interacted with TBK1/IPS-1. Further, porcine DDX3X promoted TBK1/IPS-1 to activate the IFN-P promoter in a dose-dependent manner, which further confirmed that porcine DDX3X interacted with TBK1/IPS-1.5. The interaction between porcine DDX3X and PRRSVpEGFP-C2-DDX3X was transfected into Marc-145 cells which were then infected with PRRSV. The subcellular localization of porcine DDX3X was observed by laser confocal microscopy after DAPI staining. The result made it clear that PRRSV didn’t change the porcine DDX3X’s subcellular localization. Further study found that porcine DDX3X inhibited the proliferation of PRRSV, which was detected by absolute quantification and TCID50 and PRRSV could increase the expression of porcine DDX3X mRNA in PAM cells.更多还原