【作者】 熊义；

【导师】 郑从义；

【作者基本信息】 武汉大学 ， 微生物， 2009， 博士

【摘要】 口蹄疫(Foot-and-mouth disease, FMD)是一种在全球范围内广泛传播的烈性传染病,主要感染偶蹄类动物。口蹄疫的爆发对畜牧业生产危害极大,被国际兽疫局(OIE)列为A类家畜传染病之首。口蹄疫病毒(Foot-and-mouth Disease Virus, FMDV)是口蹄疫的病原体,属于小RNA病毒科口蹄疫病毒属,为单股正链RNA病毒,基因组全长约为8500个核苷酸。和其他RNA病毒一样,FMDV基因组的多变性给对其防治带来了很大困难。因此,如何研发有效的预防和治疗FMDV感染的药物是当务之急。α-干扰素是一种广谱抗病毒药物,对FMDV的感染有一定的抑制作用。但由于FMDV能够通过阻断宿主帽子结构依赖的蛋白翻译机制来抑制α-干扰素的表达,使得通过细胞内表达α-干扰素来抗FMDV感染的效果差强人意。为了克服FMDV的这种作用机制,增强α-干扰素的抗病毒效率,本研究通过直接PCR的方法,从猪肝组织基因组中克隆了含有信号肽序列的α-干扰素基因,将其连接从FMDV克隆的IRES序列的3’端,构建了α-干扰素表达重组质粒p-IRES-IFN,使α-干扰素在细胞内的表达受到IRES的调控。同时将α-干扰素基因连接到功能完全丧失的IRES突变体的3’端,构建了一系列对照重组质粒：pc-IRES△G-IFN、pc-IRES△T-IFN,使α-干扰素在细胞内的表达受到突变的IRES的调控。将以上重组质粒转染PK细胞后发现,未感染病毒条件下,α-干扰素能够表达,受IRES的影响不大。当转染α-干扰素表达重组质粒的PK细胞被0.1 pfu/cell的水泡口炎病毒(Vesicular stomatitis virus, VSV)感染12小时后,除了转染pc-IRES-IFN的细胞中α-干扰素表达水平降低外,其他均有所增强；而在感染24小时后,所有细胞的α-干扰素表达水平都超过了感染前的水平。这说明VSV的感染不仅不能抑制内源或者外源α-干扰素基因的表达,而且会刺激内源α-干扰素基因的表达。源于FMDV的IRES在VSV感染时,对α-干扰素的表达基本没有影响。当转染α-干扰素表达重组质粒的PK细胞受到0.1 pfu/cell的FMDV感染后,受突变的IRES调控的到α-干扰素基因的表达受到抑制：在感染12小时后,表达量仅为感染前的70%左右；感染24小时后,表达量继续下降到感染前的50%左右。而受野生型FMDV IRES调控的α-干扰素基因在感染12小时后,表达量为感染前的90%左右；感染24小时后,表达量增加到感染前的125%左右。研究结果表明在IRES的调控下,α-干扰素能够克服宿主翻译抑制且持续表达。同时,通过荧光定量RT-PCR技术检测结果显示：PK细胞转染重组质粒p-IRES-IFN后,FMDV在其中的复制水平只有正常PK细胞中的复制水平的2%；PK细胞转染重组质粒pc-IRES△G-IFN和pc-IRES△T-IFN后,FMDV在其中的复制水平只有正常PK细胞中的复制水平的10%；这说明外源表达a-干扰素能够抑制FMDV的复制,IRES的调控能够增强α-干扰素的抗病毒效果。此外,噬斑试验也证实了上述结果。RNAi技术被广泛的用于抗病毒感染。以mir-30为模型,通过软件设计出潜在的能够使FMDV 3D基因沉默的特异性miRNA序列,并以pcDNA3.1(+)质粒为载体构建能够表达成熟miRNA的重组质粒p3D-1250。将p3D-1250转染BHK-21细胞,并筛选出稳定细胞系,通过感染FMDV研究miRNA对FMDV复制的干扰作用。蚀斑试验结果表明特异性miRNA可以抑制FMDV的复制,实时荧光定量RT-PCR检测数据显示特异性miRNA抑制FMDV的效率达50%,可见设计的特异性miRNA能够成功干扰FMDV的复制。更多还原

【Abstract】 Foot-and-mouth Disease (FMD) is one of the most devastating viral diseases of cloven-hooved animal species. Each breakout of FMD caused great economical damage to stock raising and international trade, so it had been classified as an OIE List A disease. Foot-and-mouth Disease Virus (FMDV) is the causative agent of FMD, belongs to Aphthovirus genus of the Picornaviridae family, it contains a signal positive-strand which is about 8400nt. Like other RNA virus, its genome variability causes a big problew for its preventing and treating. Recent studies mostly focus on how to develop efficient anti-FMDV drugs.Alpha interferon (IFN-a) is a widely-used antiviral drug. Previous studies have shown that IFN-a was able to suppress FMDV replication and spread. FMDV can also inhibit IFN-a expression in infected cells by blocking cap-dependent translation. To overcome the blockade on IFN-a mRNA translation during FMDV infection, we generated an IRES-IFN construct named pc-IRES-IFN that carries FMDV’s internal ribosome entry site (IERES) cDNA sequence between the promoter and porcine IFN-a gene. Mutant IRES was also introduced to replace the wild-type IRES as contral, there were called pc-IRES△T-IFN, pc-IRES△G-IFN. The data showed that IFN-a could express at normal situation, and had antiviral activity. We performed the infection experiments with VSV. After a 12 hour infection, PK cells transfected with pc-IRESAG-IFN and pc-IRES△T-IFN, as well as untransfected control cells, displayed no change in IFN-a production. In contrast, cells transfected with pc-IRES-IFN showed a 20% decrease in IFN-a productions. However, after 24 hours, IFN-a production in all transfected cells, as well as the untransfected control, increased to pre-infection level or above. Those demonstrate that the expression levels of IFN-αdisplayed the same trends at different timeframes. When infection by FMDV, the expression level of IFN-αunder the regulation of mutant IRES would decrease to-70% of pre-infection at 12h.p.i and -50% of pre-infection at 24h.p.i; but the expression level of IFN-αunder the regulation of wild-type IRES would decrease to-90% of pre-infection at 12h.p.i and increase to 125% at 24h.p.i. we also evaluated the antiviral activity of IFN-αby measuring the amount of viral RNA. The date of real-time RT-PCR demonstrated that the amount of viral RNA in cells transfected with pc-IRES-IFN was fivefold lower than that in cells transfected with pc-IRES△G-IFN and pc-IRES△T-IFN. Further, all of them are outclassed by the mock-transfected cells. Plaque assays were performed to confirm that cells transfected with pc-IRES-IFN were more resistant to FMDV infection than those transfected with pc-IRES△G-IFN and pc-IRES△T-IFN. It’s easy to conclude that IFN-αexpression under the regulation of FMDV IRES could provide enhanced protection to transfected cells.Today’s studies show that RNAi is a powerful antiviral tool. According to the mir-30 model, we designed a miRNA expression casstte which was named p3D-1250, targeted the 3D region of FMDV genome. BHK-21 cells were transfected with p3D-1250 and stable cells were selected by G418. Plaque assays showed that cells transfected with p3-D1250 could suppress FMDV replication. Real-time RT-PCR demonstrated that p3D-1250 could inhibite FMDV replication to 50%. All of the data indicated that designed miRNA could effetively inhitite FMDV replication.更多还原