【作者】 陈龙；

【导师】 陈焕春； 曹胜波；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2013， 硕士

【摘要】 乙型脑炎病毒(JEV)属于黄病毒科黄病毒属的成员,是一种危害严重的人畜共患病病原。该病毒严重危害人类健康,感染人后入侵中枢神经系统,造成死亡或者是留下严重的神经系统后遗症。同时它可以感染猪群,造成种猪繁殖障碍,最终导致养猪业的巨大经济损失。miRNA是基因在转录后水平调控的一种重要方式,在细胞生长、分化、细胞凋亡及代谢等生命过程中都起着重要作用。miRNA在病毒感染入侵及宿主细胞的抗病毒等方面也发挥着调节作用。而目前对于JEV诱导细胞niRNA表达谱的研究暂无报道,相关研究对于深入认识JEV的致病机制十分重要。本研究具体内容如下：将JEVP3和SA14-14-2毒株分别感染U251细胞后,提取总RNA,通过solexa测序技术,鉴定出JEV诱导U251的miRNA表达谱的变化情况,发现了其表达谱的基本特征。与对照组相比,有161个miRNA在JEV P3感染时发生了改变,175个miRNA在JEV SA14-14-2感染时发生了改变。而与SA14-14-2感染相比,P3感染诱导了76个miRNA的改变,其中60个miRNA表达上调,16个miRNA表达下调。通过实时荧光定量PCR对部分差异表达的miRNA进行了验证。在miRNA表达谱的分析中,JEV P3能够诱导U251细胞中miR-487b的显著上调。利用软件预测发现miR-487b能够靶向病毒的NS3基因,并且靶位点具有一定的保守性。进一步通过双荧光素酶报告系统检验发现miR-487b确实能够作用于NS3基因并抑制其表达,突变该位点后,抑制效果就被解除。将人工合成的miR-487b转染到细胞中后可以明显的下调病毒基因及蛋白的表达水平,抑制病毒复制,使病毒滴度明显降低。miR-487b能够有效的抑制JEV在细胞中的增殖水平,有望成为一种治疗JEV感染的手段。更多还原

【Abstract】 Japanese encephalitis virus (JEV), a member of flaviviridae, is a severe pathogen of zoonotic infectious diseases. The virus could cause serious damage to human being through invading central nervous system after infection of human being, causing the patient dead or sufferring from serious neural sequelae. At the same time JEV can infects swine, resulting in breeding obstacle, leading to significant economic losses in swine industry. As an important way of the post-transcription gene regulatory mechanism, miRNA takes part in a wide range of life processes, including growth, differentiation, apoptosis and metabolism. MiRNA also plays an important role in viral entry and the antiviral response in the host cells. But no report is available about the cellular miRNA profile induced by JEV.In this study, total RNAs from JEV P3strain and SA14-14-2strain infected U251cells were extracted and then subjected to solexa sequencing. Compared with control,161miRNAs were differentially expressed upon JEV P3strain infection while175miRNAs were differentially expressed upon JEV SA14-14-2infection. Compared with SA14-14-2infection,76miRNAs were differentially expressed upon JEV P3infection. Further validation of some of the differentially expressed miRNAs was done by real time PCR.miR-487b was up-regulated significantly upon JEV P3infection in U251cells.miR-487b was bioinformatically predicted to target NS3gene of viral genomic RNA,and the MREs were conserved to some extent.furthermore,this prediction was validated by dual luciferase reporter system.viral mRNA and proteins were down regulated when synthetic miR-487b mimics were transfected into the cells.this results indicate that miR-487b could be useful for miRNA-mediated antiviral therapeutic strategies.更多还原