【作者】 王鹏雁；

【导师】 陈创夫；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 目的:口蹄疫是由口蹄疫病毒引起的一种偶蹄动物急性、高度传播性疾病,由于口蹄疫病毒存在7个血清型和70多个亚型,各型间不存在交叉保护作用,所以用疫苗和传统的自然育种方法很难控制此病。而且此病近年来在无口蹄疫国家的爆发给发病国和邻近国家造成巨大的经济损失。因此,研究并发展新的抗病毒方法以控制口蹄疫是非常必要的。RNA干扰(RNAi)是广泛存在于生物体内的由21-23nt小双链RNA诱导序列特异性的转录后的基因表达沉默现象,RNAi对基因表达的调控、病毒感染的防护、基因转座的控制等都具有重要的意义。RNAi从发现开始就受到病毒学研究者的高度重视,被认为是抗病毒感染最有效的方法之一。许多学者以人工诱导RNAi的方式进行了病毒复制的干扰研究,并取得了良好的进展。为了发展控制口蹄疫的新方法,本研究对口蹄疫病毒的体内外复制进行了RNA干扰的探索,在此基础上,构建了靶向口蹄疫病毒的siRNA的转基因小鼠模型,并对所得的转基因鼠进行抗病毒的评价。为RNAi应用于口蹄疫病毒的基因功能研究以及口蹄疫的防治积累了必要的实验数据,为动物的抗病育种提供一条新的思路。方法:对7个血清型的FMDV基因组序列进行同源性比较后,初步筛选7个siRNA作用的靶位点。根据siRNA表达载体pSilencer 5.1–H1 Retro对插入序列的要求,设计合成了7个针对FMDV的shRNA表达序列的DNA序列。构建针对以上靶序列的siRNA表达载体。选择其中针对高保守的2B、3D区重组质粒瞬时转染BHK-21单层细胞后接种O、A、AsiaI型口蹄疫病毒检测在细胞水平上表达的siRNA对病毒复制抑制效果;将重组质粒注射乳鼠后用5 LD50和20 LD50的O型、AsiaI型FMDV感染观察小鼠死亡情况;在对细胞水平病毒繁殖有一定效果的基础上,利用显微注射法构建了靶向口蹄疫病毒2B、3D区的siRNA的转基因小鼠模型。为了检测siRNAs抗病毒的活性,我们对F1代成年鼠直接皮下接种0.5 ml 100 LD50 AsiaI FMDV口蹄疫病毒,72h后小鼠经眼眶放血处死,及时取其心脏、肝脏、脾脏、肺脏和肾脏制备常规病理切片,同时观察病变。用免疫组织化学染色法来评价转基因小鼠对口蹄疫病毒的抵抗作用。结果:经酶切和序列测定,正确构建了靶向FMDV的siRNA表达载体,分别命名为:pSi-FMDV1、pSi-FMDV2、pSi-FMDV3、pSi-FMDV4、pSi-FMDV5、pSi-FMDV6、pSi-FMDV7。用大量制备的重组质粒pSi-FMD2、pSi-FMD3转染BHK-21单层细胞后接种病毒,结果发现,与对照组相比,实验组细胞出现细胞病变现象的程度要轻;不同时间段收集的细胞上清液测得的TCID50值比对照有一定程度的降低。注射重组质粒后对小鼠攻毒可使小鼠模型发病率降低,小鼠的死亡数减少。在以上基础上将上述两个包含有整个表达框的siRNA片段经显微注射法构建了转基因小鼠模型。FMD2基因共注射506枚受精卵,移植到23只假孕母鼠输卵管内,其中17只怀孕,产仔104只。FMD3基因共注射469枚受精卵,移植到22只假孕母鼠输卵管内,其中9只怀孕,产仔55只。所有F0代小鼠经PCR检测,结果共有6只为阳性。转基因亲代小鼠与正常异性健康小鼠交配后,其后代鼠经PCR检测,有20%-30%遗传获得转基因;转基因小鼠之间进行交配后,100%的后代鼠遗传获得转基因。目前,FMD2基因传代到F3代。FMD3基因已经传至F6代,仍有20%左右转基因阳性小鼠检出。转基因小鼠外表未见任何异常。对F1代成年鼠直接感染口蹄疫病毒,感染后的转基因小鼠取组织制备石蜡切片,与正常小鼠感染后的组织切片比较病理变化的程度;同时对切片进行免疫组织化学染色,检测转基因小鼠体内表达的siRNA对病毒复制的抑制效果。结果表明:与对照相比,转基因小鼠攻毒后各组织器官病变轻微;其中,脾脏的脾小体明显增大,显示转基因小鼠体内细胞免疫功能加强;免疫组织化学结果显示,在对照组小鼠的脾脏、肝脏和肾脏中有病毒粒子的存在。而在转基因小鼠,只在脾脏和肝脏中可见少量的阳性病毒粒子。结论:以上结果表明设计的siRNA在细胞水平上和个体水平上都对口蹄疫病毒复制有一定的抑制效果;为大家畜的抗病育种提供了一个思路。更多还原

【Abstract】 Object: Foot-and-mouth disease (FMD),whose etiological agent is foot-and-mouth disease virus (FMDV), is an acute and highly contagious disease occurring in cloven-hoofed animals. FMDV has 7 serotypes and over 70 subtypes. Owing to the absence of reciprocal protection among all the serotypes, it is difficult to control FMD through vaccination and impossible to eliminate FMD by conservative natural breeding. A recent occurrence of a large epidemiogenesis has made the development of emergency antiviral strategies essential for preventing outbreaks of FMD.RNA interference(RNAi)refers to the process of sequence-specific, posttranscriptional gene silencing (PTGS) conserved in a wide range of eukaryotic organisms from plants to mammals,which is induced by 21- to 23-nucleotide (nt) siRNA that demonstrates sequence homology to the silenced gene. It is an important mechanism of cellular defense and differentiation regulation and plays an important role in the regulation of gene expression,the prevention of viral infection and the control of gene transposition. siRNA has showed its potential antiviral abilities in cultured mammalian cells and animals,and has been used as a new powerful tool for gene-specific therapeutics for viral disease. With the progressions of the research about RNAi,some investigations have been performed successfully to inhibit the replication of viruses. To develop new methods for controlling FMD, In this work, we describe the use of RNAi in inhibiting virus replication in BHK-21cells and suckling mice. On the foundation,FMDV siRNAs transgenic mice model were prepared and the resistance to replication of FMDV was evaluated, which offers an insight into the use of RNAi in animal breeding for disease resistance and necessary experimental data for study on gene function of FMDV and prevention of FMD by RNAi.Methods: Firstly,7 target sequences aimed at the FMDV mRNAs of the construction proteins were designed,the insert DNA sequences were synthesized respectively and their expression vector were constructed based on the plasmid pSilencer 5.1-H1 Hygro(Ambion).Two recombinant plasmids targetted to conservative 2B、3D regions were transfected to BHK-21 cell monolayer.The inhibition of siRNAs to FMDV replication was detected on transfected cell ,which were inoculated with O、A、AsiaI FMDV; Suckling mice were infected with 5 LD50 and 20 LD50 O、A FMDV after injected with two plasmids and the death numbers were observed; On the foundation,transgenic mice of FMDV siRNAs were prepared by microinjection. To test the anti-FMDV activity of the siRNAs, we challenged F1 positive transgenic mice with 0.5 ml 100 LD50 AsiaI FMDV by a peritoneal injection. All saline-treated mice were control group. 72h after the viral challenge,the mice were executed by exanguinating from fossa orbitalis and the heart,liver,spleen,lung,kidney were cut and fixed by 10% formaline.Routine pathological section were prepared and the pathologic change was observed by microscope. The resistance to FMDV infection of transgenic mice was evaluated by immunohistochemistry assay.Results: By identification, FMDV siRNA expression vectors were constructed and they were named: pSi-FMDV1、pSi-FMDV2、pSi-FMDV3、pSi-FMDV4、pSi-FMDV5、pSi-FMDV6、pSi-FMDV7. It was demonstrated that transfection of BHK-21 cells with the 2 siRNA-expressing plasmids --pSi-FMD2、pSi-FMD3-- could induce a lower CPE compared with the controls. Further, the TCID50 of the FMDV serotypes A, O, and Asia 1 detected in supernatants collected from cells transfected with FMDV-specific siRNA-expressing plasmids was lower than that of control cells. In addition, when challenged by 5 LD50 or 20 LD50 of the FMDV serotypes A, O, or Asia 1 after injecting FMDV-specific siRNA-expressing plasmids, 10–40% suckling mice could resist virus infection. On the foundation,transgenic mice of FMDV siRNAs were prepared by microinjection.The positive integration rate of F0 was low,while that of F1、F2、F3、F4 were 10%-20%.At present F6 transgenic mice were passaged and the phenotype of offspring had no any abnormality. From the results of H.E staining of different pathological section,compared with control,the pathologic change of transgenic mice was light or almost normal.Especially, the splenic corpuscle of transgenic mice augmented and macrophage、epithelioid cell node in junction of white pulp and red pulp increased obviously.The results of immunohistochemistry showed that FMDV could be detected in liver,spleen,kidney of control mice and the number of virus in spleen was large.However,FMDV only could be detected in spleen and liver of transgenic mice and the number was little,compared with control.Conclusion: The results showed that the siRNAs targetted to FMDV can inhibit virus replication in vitro and in vivo at some degree,which offered an insight into the use of RNAi in large livestock breeding for disease resistance.更多还原