【作者】 刘伟；

【导师】 许修宏； 王笑梅；

【作者基本信息】 东北农业大学 ， 微生物学， 2007， 硕士

【摘要】 鸡传染性法氏囊病(Infectious Bursal Disease，IBD)是危害雏鸡的一种急性、高度接触性、病毒性传染病，给世界范围的养鸡业造成巨大经济损失。病原为鸡传染性法氏囊病病毒(Infectious Bursal Disease Virus IBDV)，IBDV对宿主淋巴细胞有严格嗜性，其RNA基因组易变异，常规的疫苗设计策略难以产生理想的免疫效果，因此，探索防治该病的新方法有重要意义。RNA干扰(RNA interference，RNAi)作为新的有效抗病毒工具，其机制与建立在病毒蛋白基础上的传统的疫苗免疫机制不同，是转录后mRNA水平上的基因沉默机制，通过双链RNA(double-stranded RNA，dsRNA)分子阻断或者降低同源基因的表达而达到抗病毒复制的效果。在哺乳动物细胞中，21～23nt的小干扰dsRNA(small interfering RNA，siRNA)可以诱导同源基因特异表达抑制，为探索抗病毒研究提供了新思路。本研究针对IBDV中RNA聚合酶(VP1)的编码基因、主要结构蛋白VP2、VP3的编码基因，使用http://www.ambion.com的靶位点筛选和设计工具，分别选取3个特异siRNA序列(VP1：siRNA618、siRNA1115和siRNA2571，VP2：siRNA989、siRNA1143和siRNA1311，VP3：siRNA222、siRNA314和siRNA448)，同时设计无相关序列(siRNA-CON)对照，利用体外转录法产生siRNA。VP1-siRNA、VP2-siRNA、VP3-siRNA转染Vero细胞后感染IBDV，通过病毒蚀斑和荧光定量PCR法检测VP1-siRNA对IBDV复制的抑制效果。VP2-siRNA、VP3-siRNA与表达绿色荧光蛋白(Green fluorescent protein，GFP)的重组质粒pEGFP-VP2、pEGFP-VP3共转染Vero细胞，荧光显微镜下检测表达GFP蛋白细胞比例，快速筛选有效VP2-siRNA、VP3-siRNA片段，通过病毒蚀斑和流式细胞仪分析抑制效果，验证了筛选片段的基因沉默效果。本研究为RNAi技术应用于抗IBDV感染研究奠定了基础并为研究病毒蛋白功能提供了新途径。更多还原

【Abstract】 Infectious Bursal Disease (IBD) is an acute, high contact, virulent infectious disease. It is oneof the most important causes of economic loss to poultry industry worldwide. The etiological agentis Infectious Bursal Disease Virus (IBDV). IBDV has so strictly tropism to the host lymphocyte andhas so variable genome that it is hard for the traditional vaccine design strategies to have the idealimmunity effect. Therefore, exploring the new preventing and controlling method has the vitalsignificance.As a new effective antiviral tool, RNA interference (RNAi) has a different mechanism fromclassic vaccine which is based on the immunogenicity of proteins. It is a post-transcriptionalmechanism of sequence-specific gene silencing that initiated by double-stranded RNA (dsRNA). Inmammalian cells, short interfering RNA (siRNA) (21-25nt) can specifically silence the expressionof the corresponding gene. It provides a new method in anti-virus research fields.This research in view of IBDV in RNA polymerase (VP1) the coding gene, main structureprotein VP2, the VP3 coding gene, used http://www.ambion.com the target site screening and thedesign tool, separately selected 3 specific siRNA sequence (VP1:siRNA618, siRNA1115 andsiRNA2571, VP2: siRNA989, siRNA1143 and siRNA1311, VP3: siRNA222, siRNA324 andsiRNA448). Simultaneously a non-specific siRNA sequence (siRNA-CON) was designed asnon-specfic control. These siRNAs were synthesized by transcription in vitro.In this study, Vero cells were infected by IBDV after being transfected with these siRNA byliposomes. To research the inhibition effect of VP1-siRNA, VP2-siRNA, VP3-siRNA, real-timePCR was used to detect the synthesis of IBDV RNA and virus titration was measured by PFU(Plague-forming unit). VP2-siRNA, VP3-siRNA were transfected with recombinating plasmidpEGFP-VP2, pEGFP- VP3 that expressing Green fluorescent protein (GFP) into Vero cells. Theratio of expressing Green fluorescent protein cells were detected by fluorescent microscope. Thismethod was used to screen rapidly the effective VP2-siRNA, VP3-siRNA. Eventually, the real-timePCR, PFU and flow cytometry were used to detect the inhibiting result confirmed gene silencingresult.This study provided a primary material for anti-IBDV infection by RNAi and provided a newapproach to study of the function of viral proteins in IBDV infection.更多还原