【作者】 倪伟；

【导师】 陈创夫； 乔军；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 牛病毒性腹泻病毒（bovineviraldiarrheaviruses，BVDV）是牛病毒性腹泻-粘膜病的主要抗原，该病在世界各国都有比较普遍的发生和存在。该病对我国养殖业同样构成严重危胁。因此，国内、外对BVD的防治都非常关注。但是目前针对BVDV感染尚无特异性的预防疫苗、治疗方法和抗病毒药物。因此建立新的抗病毒方法，控制病毒的扩散具有重要意义。利用转基因技术进行抗病育种已成为研究热点，而抗病候选基因的克隆和验证是转基因新品种培育中急待解决的关键问题。本研究从两个角度开展抗BVDV功能基因研究：（1）靶向病毒基因设计siRNA，抑制BVDV增殖为目的，将RNAi技术与转基因技术相结合，进行抗病毒功能基因研究，并在绵羊个体进行抗BVDV效果评价，为制备抗BVDV转基因家畜动物奠定基础；（2）以修饰宿主抗BVDV功能基因，提高BVDV易感动物的抗病性能为目的，通过microRNAs（miRNAs）的差异表达分析，筛选宿主抗BVDV功能基因，为我国抗病转基因新品种培育和产业化提供基因资源。首先，设计靶向BVD病毒Npro、C、NS3、NS4B、NS5A基因的shRNA干扰片段，构建相应的shRNA干扰载体pu6-sh1…pu6-sh9，分别转染MDBK细胞，48h后接种BVDV，Real-timePCR与病毒滴定测定的方法并用，测定干扰载体的抗病毒效率。结果显示pu6-sh3、pu6-sh8的干扰效果较好，最高干扰效率可达88.5%。为进一步提高干扰效率，应对病毒变异而产生的逃逸现象，将sh3、sh8的表达框连接到同一载体上，结果显示干扰效率可以提高到92.6%。将sh3-sh8表达框连接到转基因表达载体，构建靶向BVDVshRNA转基因载体pNeo-2loxp-u6-shBVDV，转染绵羊胎儿成纤维细胞，筛选稳定表达sh3-sh8基因框的细胞克隆，经PCR鉴定后，共获得阳性细胞克隆12株，经Real-timePCR与病毒滴定测定，有5株阳性细胞克隆抗病毒效果较好，可用于核移植的供体。供体细胞注入体外成熟培养的去核卵母细胞中，电融合后进行体外培养，2-8细胞期的胚胎进行胚胎移植。卵母细胞平均成熟率可以达到77.24±0.52%，重构胚的平均卵裂率为68.12±0.41%，先后共将589枚不同发育阶段的克隆胚胎移植到27只受体绵羊体内。2011年移植的12只绵羊中，获得一只流产胎儿，PCR鉴定结果呈阳性。分离流产胎儿皮肤成纤维细胞和肾上皮细胞，接种牛病毒性腹泻病毒NADL株和C24V株，经Real-timePCR与病毒滴定测定，均有良好的抗BVDV的效果。这部分研究为进行抗BVDV转基因动物探索奠定基础。为了探讨宿主细胞抗BVDV机制，筛选影响病毒释放的宿主抗病毒功能基因，本研究通过高通量测序技术获得BVDV感染与未感染的MDBK细胞miRNAs差异表达谱。miRNAs差异表达分析结果显示：BVDV感染的细胞与正常细胞相比，26个miRNAs表达量显著上调，2个miRNAs表达量显著下调，生物信息学预测结果显示miR-1249与细胞凋亡密切相关。Hoechst染色、流式细胞仪检测、caspase酶活性检测结果显示，miR-1249可促进细胞凋亡。WB、荧光素酶报告基因检测系统分析结果显示miR-1249可以靶向抑制Faim2蛋白的表达。揭示miR-1249通过抑制死亡受体凋亡通路中Faim2蛋白的表达促进细胞凋亡、病毒释放，有利于宿主细胞清除病毒感染，为从提高宿主自身抗病毒能力角度出发，研究抗BVDV转基因育种材料提供科学理的论依据。更多还原

【Abstract】 Bovine viral diarrhea virus (BVDV) is an important animal pathogen which results in bovine viral diarrhea-mucosal disease. BVDV is a global pathogen of cattle. BVDV occurs worldwide and despite the development of different vaccines and eradication programs, it still causes pronounced economic losses in the cattle industry. Therefore, it is very important to establish a new anti-virus method for controling BVDV. Our study will do preliminary studies on evaluating antiviral gene against BVDV from two aspects.(1) To investigate the inhibition effect of shRNAs on virus replication, transgenic sheep were developed by SCNT and RNAi technology.(2) To screen anti-BVDV functional genes of host animal, expression profiles of microRNAs were constructed, which will provide genetic resources for cultivating new resistant transgenic animal.We designed9shRNAs targeting NPro、C、NS3、NS4B、NS5A region of BVDV respectively. These shRNAs were cloned into pGenesil-1for assessing antivirus activity. The effect of RNAi on NADL replication was further examined by using real-time RT-PCR and virus titers at48h after virus challenge. The pU6-sh3and pU6-sh8markedly reduced expression of viral RNA by88.5%and85%, respectively, compared to control group. In order to further improve the RNAi efficiency and prevent the virus escape the phenomenon, the the sh3and sh8were expressed in the same vector. The results showed that the RNAi efficiency can be improved to92.6%. The transgenic expression vector (pNeo-21oxp-u6-shBVDV) with sh3-sh8expression box were constructed and transfected in sheep fetus fibroblasts. After transfection, cells were subjected to G418screening.12positive cell clones were obtained by selection and identified by PCR.5positive cell clones preferably antiviral effect as determined by the real-timePCR and virus titration. These cells can be used in nuclear transplantation as donor cells. Donor cells were injected in vitro maturated oocyte and then electric fusion.2-8cell stage embryos were used for embryo transfer. The average mature oocytes rate was up to77.24±0.52%, cleavage rate of reconstructed embryo was up to68.12±0.41%. A total of589cloned embryos at different developmental stages were transplanted into27receptor sheep. An aborted fetus were obtained from receptor sheep. The fetus were transgenic as identificated by PCR. Skin fibroblasts and kidney epithelial cells were isolated, cultured and inoculated with bovine viral diarrhea virus NADL strain C24V strain. These cells showed a good anti-BVDV effect as determinated by Real-time PCR and virus titration assay.In order to study anti-virus mechanism of host cells and screen anti-BVDV functional gene of host animal. In this study, microRNA expression profiles were obtained by high-throughput sequencing technology from BVDV-infected and-uninfected MDBK cells. Differential expression analysis of microRNA showed that26microRNA expression levels significantly raised,2microRNA expression levels significantly reduced in BVDV-infected cells when compared with BVDV-uninfected cells. Hoechst staining, flow cytometry and caspase activity detection were used to determinated the role of miRNA. Our results showed that miR-1249can promote apoptosis. Western blotting and luciferase reporter system analysis showed that miR-1249can be targeted inhibit Faim2protein expression. Our results showed that miR-1249may promote apoptosis and virus release by inhibiting the expression of death receptor apoptotic pathway Faim2protein. Our results provide a scientific theory for cultivating new resistant transgenic animal by enhancing the anti-virus ability of BVDV-susceptible animal更多还原