【作者】 张玉颖；

【导师】 吴润； 刘光清；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 应用反向遗传系统实现RNA病毒的拯救,从而可以在DNA水平上对RNA病毒进行遗传操作,为深入研究RNA病毒的分子生物学及病毒与宿主的相互作用提供了强有力的技术工具。对RNA病毒进行遗传拯救或构建感染性克隆己成为分子病毒学实验室进行病毒结构与功能深入研究的必经之路。兔出血症(RHD)是由兔出血症病毒(RHDV)引起的兔的一种急性﹑败血性﹑高度致死性传染病,是危害世界养兔业的严重传染病之一。本研究选择兔出血症病毒JX/97分离株,克隆其全基因组cDNA,在国内首次尝试构建RHDV反向遗传系统,拯救经过分子标记的RHDV。1.RHDV JX/CHA/97株全长cDNA分子克隆的构建设计并合成了覆盖JX/CHA/97株基因组的5对引物,从人工感染RHDV致死的兔肝组织中提取病毒总RNA,采用RT-PCR方法分别扩增各基因片段。通过沉默突变引入一分子标记(EcoR V识别位点)以便于将来区分自然毒株和基因工程毒。利用限制性内切酶,分别消化各基因扩增片段和pBluescript II SK(+)(即pSK)载体,构建JX/97株基因组的全长cDNA分子克隆(pBlRHDV)。分别采用PCR法﹑限制性内切酶酶切法和基因组测序法对pBlRHDV进行鉴定,结果表明已成功构建了RHDV JX/97株的全长cDNA分子克隆。它不仅含有JX/97株的全基因组序列,还含有SP6启动子序列﹑一个分子标记(EcoR V识别位点)和引物设计中添进的限制性酶切位点。2. RHDV JX/CHA/97株全长cDNA分子克隆(pBlRHDV)感染性的鉴定用Nru I将pBlRHDV线形化后,以之做模板,使用RiboMAXTM Large Scale RNA Production Systems-SP6聚合酶系统进行体外转录,得到病毒RNA。用脂质体转染法将转录物导入RK-13细胞,24h后可观察到典型的RHDV致细胞病变效应。分别使用RT-PCR、皮下和腹腔接种SPF健康兔以及免疫电镜观察等方法进行鉴定,结果表明拯救病毒成功,获得了感染性cDNA分子克隆。通过对感染性分子克隆分子标志的鉴定,排除了自然毒株的污染。总之,RHDV反向遗传研究系统的建立将为深入研究其功能基因组学以及致病和变异的分子机制奠定基础,同时还具备研制新一代基因缺失疫苗和标记疫苗的潜在应用前景。更多还原

【Abstract】 Using reverse genetics systems for RNA virus rescue and manipulating in DNA level is a strong technique to study molecular biology and interactions between RNA virus and host. Genetic rescue or constructed infectious clone of RNA virus become the necessary way to study structure and function of virus in molecular virology laboratory. Rabbit hemorrhagic disease is a kind of acute﹑lethal infectious disease caused by rabbit hemorrhagic disease virus and have a great harm to rabbit production all over the world. This study selected rabbit hemorrhagic disease virus which was the pathogen of Rabbit hemorrhagic disease as a model on the basis of obtainment of full-length cDNA clone of RHDV JX/97 strain and attempt to construct RHDV reverse genetics systems and rescue molecular marked RHDV in our country firstly.1. Construction of full-length genome cDNA clone of RHDV JX/CHA/97 strainFive pairs of primers were designed for sequencing the genome of JX/CHA/97.Strain virus total RNA was extracted from rabbit liver tissue died from RHDV infected and each sequence was obtained by RT-PCR .A molecular marker (EcoR V enzyme site) was introduced to distinguish wild-type virus and rescue virus. Each gene amplified fragments and pBluescript II SK(+) vector were digested by restriction enzyme to construct full-length genome cDNA(pBlRHDV)clone of JX/97 strain. The pBlRHDV was identified by PCR﹑restriction enzyme digested and genome sequenced. The results showed that full-length cDNA clone of RHDV JX/97 strain was constructed successfully. The whole genome sequence﹑SP6 promoter sequence﹑a molecular marker(EcoR V enzyme site) and restriction enzyme digestion site added which primer designed were all found in pBlRHDV clone.2.Identification of infection of full-length cDNA clone(pBlRHDV)of RHDV JX/CHA/97 strainpBlRHDV used as a template after linearized with Nru I and virus RNA synthesized in vitro by means of RiboMAXTM Large Scale RNA Production Systems- SP6 polymerase system . RK-13 cell was transfected with transcribed product in vitro using lipofectamine reagent, typical RHDV CPE would be seen after 24 hour. RT-PCR﹑inoculated subcutaneousely and intraperitoneally to unvaccinated sensitive healthy rabbit and immunosorbent electron microscope observation was used for detection. All the detection results showed rescued virus was obtained and constructed infectious cDNA clone of RHDV successfully. The infection of wild-type virus was excluded through detection of molecular marker of infectious clone.In a word, RHDV reverse genetics can be used to study functional genomics, molecular mechanism of pathogenesis and variation of RHDV and show some prospects in research of gene-deleted vaccine and marker vaccine.更多还原