抗体介导的siRNA靶向运输并抑制汉坦病毒复制的研究

发布时间：2018-10-20 07:04

【摘要】： 汉坦病毒是脂质被膜包裹的RNA病毒,可以持续感染啮齿类动物却不使其致病,而人在吸入含有病毒的气溶胶状的啮齿动物排泄物后可以被感染,引起肾综合征出血热(HFRS)和肺综合征(HPS)。目前,还没有针对汉坦病毒感染治疗的抗病毒药物,临床只限于对症和支持治疗。因此,寻找有效和特异的治疗汉坦病毒的新方法显得十分重要而迫切。 RNA干扰(RNAi)是一种由双链RNA诱导的序列特异性的基因沉默机制。以致病基因为靶点的RNAi技术,可用于治疗包括肿瘤、感染性疾病和遗传病在内的各种疾病。抗病毒RNAi策略作为治疗方法的应用研究正备受关注,有多种基于该策略的方案正在进行临床试验。然而,尽管针对病毒基因的RNAi能够特异性地抑制病毒的复制,但小RNA的组织或细胞特异性转运却绝非简单易行。治疗性小干扰RNAs(siRNAs)面临着如何在体内被特异和有效地转运到靶细胞的难题。研究结果提示,体内细胞类型特异性的基因沉默可以通过siRNA与细胞类型特异性亲和配体或单克隆抗体结合而得以实现,即抗体指导的siRNA复合物通过内吞作用进入靶细胞,随后释放到细胞浆,通过RNAi路径,特异性地沉默靶基因的表达。与小分子碱性核酸结合蛋白融合的抗体片段是一种体内转运的有效工具,体内基因沉默的特异性、不引发非特异性免疫激活和全身毒性等特点促进了以治疗性RNAi的细胞类型特异性转运为基础的治疗方法的开发。本研究针对汉坦病毒76-118株的编码基因,以随机序列siRNA为阴性对照,设计合成了4种siRNAs,筛选出抗病毒效果最强的siRNA-HV2(命名为siHV)用于下一步研究。为确定抗体介导的siRNA是否可以特异性地将siRNA转运入汉坦病毒感染的细胞,在原核表达载体pET32a中表达了一种融合蛋白3G1scFv-C_K-tP,它由汉坦病毒抗原特异性单链抗体(3G1scFv)与鱼精蛋白片段(第8-29位氨基酸)通过人C_K连接而形成。诱导表达的此融合蛋白以可溶性表达为主,此融合蛋白明显保留有抗原结合活性,可以特异地结合胞膜上的病毒抗原,与细胞表面的抗原结合后可以特异性地内化入感染细胞的胞浆,且与转铁蛋白有相似的内化路径,即通过网格蛋白介导的内吞来实现的。此融合蛋白可以结合并转运siRNAs到汉坦病毒感染的细胞。先后用凝胶迁移阻滞实验和流式细胞仪进行检测,结果表明:3G1scFv-C_K-tP具有与siRNA结合的能力,且这种结合能力存在着剂量依赖关系;DynabeadsTALON磁珠分析显示3G1scFv-C_K-tP与siRNA之间量的结合关系为1分子3G1scFv-C_K-tP可以结合约10分子siRNA;3G1scFv-C_K-tP可以将FITC-siRNA特异性地运送到病毒感染的细胞。采用MTT方法测定细胞生长曲线,表明3G1scFv-C_K-tP/siRNA对细胞生长没有毒性作用。在感染细胞中,此融合蛋白靶向转运siHV能够有效地抑制病毒的复制。结果表明:3G1scFv-C_K-tP/siHV降低了病毒RNA和病毒抗原的水平,而且下降程度与siRNA呈剂量依赖性。在乳鼠颅内注射汉坦病毒76-118株以制备动物模型,然后腹腔注射3G1scFv-C_K-tP/FITC-siRNA混合物,证实此融合蛋白能够特异地将siRNA转运入汉坦病毒感染的脑细胞。流式细胞仪检测显示3G1scFv-C_K-tP可以特异性的将FITC-siRNA转运到汉坦病毒感染的脑组织,且荧光显微镜下可见荧光阳性细胞、高倍镜下观察到荧光弥散分布在汉坦病毒感染细胞的细胞膜和细胞浆。腹腔注射3G1scFv-C_K-tP/siHV可以用于汉坦病毒感染性脑炎的保护。结果显示3G1scFv-C_K-tP/siHV治疗组中病毒RNA和病毒抗原的水平明显降低;将脑组织和其它组织分别进行免疫组织化学染色,观察到3G1scFv-C_K-tP/siHV使汉坦病毒的感染和复制受到了明显抑制;对动物的生存率进行评估,发现3G1scFv-C_K-tP转运的siHV对动物有明显的保护作用;检测乳鼠体内IFN水平,显示3G1scFv-C_K-tP/siHV并未诱导IFN的产生,证明在动物体内产生的保护作用是由针对病毒RNA的特异性RNAi所介导。总之,实验结果显示:3G1scFv-C_K-tP可以特异性转运siRNA,并沉默靶基因的表达,因此可以用于汉坦病毒感染的保护。
[Abstract]:Hantavirus is a lipid-coated RNA virus that can be continuously infected without causing it to cause disease, whereas humans can be infected after inhalation of a virus-containing aerosol-like rodent excreta, causing hemorrhagic fever with renal syndrome (HFRS) and lung syndrome (HPS). Currently, there are no antiviral drugs for the treatment of Hantavirus infection, which is limited to symptomatic and supportive treatment. Therefore, it is very important and urgent to find effective and specific new methods for treating Hantavirus. RNA interference (RNAi) is a sequence-specific gene sink induced by double-stranded RNA The RNAi technology, targeting pathogenic genes, can be used to treat diseases, including tumors, infectious diseases, and parasitic diseases. The application of anti-virus RNAi strategy as a treatment method is attracting more and more attention, and there are a variety of protocols based on this strategy. However, although RNAi against viral genes can specifically inhibit viral replication, tissue or cell-specific transport of small RNAs is by no means simple Single-easy. Therapeutic small interfering RNAs (siRNAs) are faced with how to be specifically and effectively transported in vivo The results suggest that in vivo cell type-specific gene silencing can be achieved by binding siRNA to cell type specific affinity ligands or monoclonal antibodies, i.e., antibody directed siRNA complexes enter the target cells by endocytic action, Over-RNAi pathway, specific sinking An antibody fragment fused with a small molecule alkaline nucleic acid binding protein is an effective tool for in vivo transport, Due to the specificity of silencing, non-specific immune activation and systemic toxicity, the specific transport of cell types with therapeutic RNAi is promoted. In this study, four siRNAs were synthesized and the strongest anti-viral siRNA-HV2 (named s) was screened. iHV) is used for the next study. To determine whether an antibody-mediated siRNA can specifically transport siRNA into Hantavirus infected cells, a fusion egg is expressed in a prokaryotic expression vector pET32a White 3G1scFv-C _ K-tP, consisting of Hantavirus antigen specific single chain antibody (3G1scFv) and protamine fragment (8th to 29th) The fusion protein is mainly expressed as soluble expression, and the fusion protein is obviously retained with antigen binding activity, can specifically bind the virus antigen on the cell membrane, and can be combined with the antigen of the cell surface and can so as to specifically internalize the cytoplasm of the infected cells and have a similar internalization path to the transferrin, i.e. This fusion protein can be combined The results showed that 3G1scFv-C _ K-tP had the ability to bind with siRNA, and the binding ability existed in dose-dependent relation; DynabeadsTAALON beads analysis showed that the binding relationship between 3G1scFv-C _ K-tP and siRNA was 1 molecule 3G1scFv-C. _ K-tP can bind about 10 molecular siRNA; 3G1scFv-C _ K-tP can bind FITC-The cell growth curve was measured by MTT assay, indicating 3G1scFv-C _ K-tP/ siRNA has no toxic effect on cell growth. In infected cells, this fusion The results showed that 3G1scFv-C _ K-tP/ siHV reduced viral RNA and virus resistance. An animal model was prepared by intraperitoneal injection of Hantavirus 76-118 strain, and then the 3G1scFv-C _ K-tP/ FITC-siRNA mixture was injected intraperitoneally to confirm the fusion. The fusion protein can specifically transport the siRNA into the brain cells infected with Hantavirus, and the flow cytometry detects that 3G1scFv-C _ K-tP can specifically transport FITC-siRNA to the brain tissue of Hantavirus infection, The fluorescence dispersion was observed in the cell membrane and cytoplasm of Hantavirus infection cells. The intraperitoneal injection of 3G1s CFv-C _ K-tP/ siHV could be used in the protection of Hantavirus infection encephalitis. The results showed that the levels of viral RNA and viral antigen in 3G1scFv-C _ K-tP/ siHV treatment group were significantly decreased, and immunohistochemical staining of brain tissue and other tissues was performed to observe 3G1scFv-C _ K-tP/ siH. V has been shown to inhibit the infection and replication of Hantavirus; assess the survival rate of animals; find that 3G1scFv-C _ K-tP trans-transporter siHV has a significant protective effect on animals; detect IFN levels in milk rats, and show that 3G1scFv-C _ K-tP/ siHV does not induce IFN production and is proved to be The protective effects produced in animals are mediated by specific RNAi targeting viral RNA. In conclusion, the results show that 3G1scFv-C _ K-tP can be specifically transported.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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