摘要
FMD(Foot-and-Mouth Disease ,FMD)是严重威胁世界畜牧业经济及人类健康的烈性传染病,是牛、羊、猪等主要畜种的头号杀手。该病病原FMDV(Foot-and-Mouth Disease virus,FMDV)属于小RNA病毒,具有高度的变异性和广泛的宿主谱,关于其致病的分子机制、抗原变异的分子机理和宿主嗜性,至今还不是十分清楚。为了研究其致病机制、抗原变异的分子机理和宿主嗜性,我们首次构建了FMDV牛源泛亚型O/CHINA/99株的3株不同感染性cDNA分子克隆。
1. FMDV O/CHINA/99株全长cDNA分子克隆的构建和感染性鉴定
设计并合成了覆盖O/CHINA/99株基因组的9条引物,从感染牛源O/CHINA/99株的乳鼠胴体中提取病毒基因组RNA,采用RT-PCR方法分别扩增出4条目的基因片段(S、C、Z和E)。利用片段两端的限制性内切酶位点,将各基因片段分别插入到pOK12载体中,构建成O/CHINA/99株基因组的全长cDNA分子克隆。用脂质体转染法将转录物RNA导入BHK-21细胞,24h后可观察到典型的FMDV致细胞病变效应。分别使用RT-PCR法和乳鼠皮内接种法对拯救的病毒进行鉴定,结果表明拯救到了特定的FMDV。以上鉴定结果表明,我们已成功构建了O/CHINA/99株全长感染性cDNA分子克隆,并拯救出基因工程病毒。这将为研究FMDV基因组的结构与功能、探讨PMDV的分子致病机制以及宿主嗜性奠定了良好的基础。
2. FMDV O/CHINA/99缺失毒株的全长cDNA分子克隆的构建和感染性鉴定
关于PKs的功能目前尚不太清楚,有报道称PKs与FMDV的宿主嗜性和毒力有一定的关系。为揭示FMDV翻译、分子致病机制以及阐明PKs基因缺失导致的病毒宿主嗜性改变的原因,我们设计并合成了O/CHINA/99株基因组5条引物,利用RT-PCR扩增各基因片段,成功构建0/CHINA/99缺失毒株全长cDNA分子克隆。并拯救出0/CHINA/99特定缺失的基因工程病毒,这为进一步探索FMDV致病的分子机制及宿主嗜性奠定了良好的基础。
3.嵌合病毒pOKTAW97/CHINA99的全长cDNA分子克隆的构建和感染性鉴定
IRES是蛋白翻译顺式作用元件,二级结构有4个结构域,IRES不依赖帽结构指导病毒蛋白合成。由于IRES的空间结构发生变化,而结合的宿主真核翻译起始因子不同,所以IRES在病毒翻译和宿主嗜性中发挥很重要的作用。为揭示FMDV蛋白翻译、分子致病机制以及阐明IRES空间结构改变导致的病毒宿主嗜性改变的原因,我们以牛源FMDV O/CHINA/99株的感染性cDNA为骨架,用猪源FMDV的IRES置换相应的区段,构建了FMDV型内嵌合病毒pOKTAW97/CHINA99的全长cDNA分子克隆,并得到置换IRES的嵌合病毒。这将为研究FMDV基因组的IRES的结构与功能、探讨FMDV的分子致病机制以及宿主嗜性创造了良好的条件。
Foot-and-mouth disease, one of the most important disease of livestock, is caused by a small RNA virus of the family Picornavh'idae. FMDV showed high variability, host tropism and the mechanism of its molecular pathogenesis hasn't been clarified. In order to research high variability and host tropism,we sequenced the full length genome of FMDV O/CHINA/99 strain isolated from cattle, and generated an infectious cDNA clone of the strain.
1. To construct the full-length cDNA of FMDV O/CHINA/99 strain, we design four pairs of over-lapping primers, which cover the whole genome of FMDV O/CHINA/99 strain, according to the nueleotide sequence of O/CHINA/99 strain. After extracting the total RNA of virus from the infected newborn mice, four cDNA fragments (S, C, Z and E) of O/CHINA/99 strain were amplified by reverse transcription PCR. These PCR products and pOK12 vector were digested with the unique restriction enzymes respectively, and ligated into pOK12 vector. Finally, the full-length cDNA of O/CHINA/99 strain was constructed. RNA synthesized in vitro by means of a T7 promoter inserted in front of the cDNA lead to the production of infectious particles upon transfection of BHK-2I cells, as shown by eytopathic effects.The rescued Virus was also found to be highly pathogenic for mice by intradormal injection producing afatal disease indistinguishable from that of wild-type virus. A full-length cDNA clone of a FMDV isolated from swine was assembled in the plasmid vector pOK12.
2. FMDV change of the host tropic,lack strains most likely to cause the host tropic change. PKs on the function is still not clear, it is reported that the PKs with the FMDV host tropic and toxicity of a certain relationship. To reveal FMDV virus translation, molecular pathogenesis and clarify PKs gene deletion led to the virus host tropic the causes of change, we design and synthesis type O FMDV behalf of Pan-Asian strain O/CHINA/99 Genome five primer. The results showed that the full-length cDNA molecule of FMDV O/CHINA/99 strain was construeted successfully. The results showed that by the O/CHINA/99 deletion of genetic engineering virus, FMDV to further explore the molecular mechanism of pathogenesis and host tropic laid a good foundation.
3. IRES is the translation of cis-element and have two of four Central domain. IRES guidance virus protein synthesis that don't rely on the structure hat. As IRES changes in the structure, and the combination of host different eukaryotic translation initiation factor, so IRES play an important role in the virus translation and host tropic.To reveal FMDV virus protein translation, molecular pathogenesis and clarify IRES spatial structure change in the host tropic virus the causes of change, we constructed a virus embedded FMDV pOKTAW97/CHINA99 the full-length cDNA molecular cloning and transcription by the virus RNA, using liposomes transfer method transcription of RNA into BHK-21 cells, by Replacement IRES the chimeric virus. This will study the IRES FMDV genome structure and function of PMDV the molecular pathogenesis and host tropic laid the foundation.
引文
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