siRNA对SARS冠状病毒复制的抑制作用研究

发布时间：2018-04-30 08:30

本文选题：SARS冠状病毒 + RNA干扰　；参考：《中国人民解放军军事医学科学院》2005年硕士论文

【摘要】：严重急性呼吸综合征(Severe Acute Respiratory Syndrome,SARS)是人类在21世纪初遭遇的烈性传染病之一,我国为该病的重要疫区。其病原体为SARS冠状病毒(SARS Coronavirus,SARS-CoV)。目前对其尚无特效的治疗方法,因而探索有效的抗SARS病毒新途径势在必行。 RNA干扰(RNA interference,RNAi)是基于RNA水平的抗病毒策略,凭借自身的特异、高效、快速等优势,它已成为抗病毒领域的研究热点之一。RNAi的发现和应用为治疗SARS病毒感染提供了新思路。本研究拟开展以下两方面内容,观察siRNA对SARS病毒复制的抑制作用。探索抗SARS病毒感染的新途径。一.siRNA的设计合成及其对SARS病毒部分复制酶基因的抑制作用由于复制酶基因的表达是SARS病毒启动转录的前提,而刺突蛋白在病毒入侵宿主细胞的过程中发挥着重要作用,因此选择SARS病毒复制酶基因(rep)和刺突蛋白基因(s)的保守序列作为靶基因,设计并合成9种siRNA(pS01～09),其中pS01～07针对rep基因,pS08～09针对s基因。由于体外合成的siRNA不易操作且基因干涉作用持续时间短,故采用构建siRNA表达载体的方法在体内合成siRNA。同时,构建融合表达绿色荧光蛋白(GFP)基因和部分rep基因的重组载体,将已构建好的识别rep基因的siRNA表达载体与重组载体共转染293T细胞,通过荧光显微镜观察和流式细胞仪分析融合基因的表达情况。结果表明,针对SARS病毒rep基因第3504nt-3522nt的siRNA可使rep基因的表达量下降33%,说明siRNA可特异而有效地抑制基因的表达。二.siRNA在Vero细胞中的抗SARS病毒作用为进一步研究siRNA对SARS病毒复制的抑制作用,将构建的siRNA表达载体分别转染Vero细胞,获得稳定转录siRNA的克隆细胞株V/pS01、03、05～09及对照细胞株V/pSN,然后以BJ01株病毒悬液感染克隆细胞,通过病毒滴度测定和间接免疫荧光实验观察siRNA对SARS病毒复制的抑制作用。结果表明,针对rep基因和s基因的siRNA(pS05和pS08)呈现出了良好的抗病毒效果,在接种SARS病毒后均未检测到病毒的特异性抗原,且病毒滴度下降10~3～10~4倍。另外,通过实时定量RT-PCR检测病毒感染的细胞上清中rep基因的含量,结果显示,细胞株V/pS05中rep
[Abstract]:Severe Acute Respiratory Syndrome ( SARS ) is one of the severe infectious diseases in the early 21st century . Our country is an important epidemic area of the disease . The pathogen is SARS coronavirus ( SARS - CoV ) . At present , there is no special treatment method , so it is imperative to explore the effective way to resist SARS virus .

RNA interference ( RNAi ) is an antiviral strategy based on RNA level . It has become one of the hot spots in the field of anti - virus by virtue of its specific , high - efficiency , rapid and so on . The discovery and application of RNAi provide a new idea for the treatment of SARS virus infection .

This study intends to investigate the inhibitory effect of siRNA on SARS virus replication , and explore the new way of anti - SARS virus infection .

1 . Design and synthesis of siRNA and its inhibitory effect on SARS virus partial replicase gene

Since the expression of the replicase gene is a prerequisite for the initiation and transcription of SARS virus , the spike protein plays an important role in the process of virus invading host cell , and 9 kinds of siRNA are selected and synthesized as the target gene by selecting the conserved sequence of the SARS virus replication enzyme gene ( rep ) and the spike protein gene ( s ) as the target gene . The siRNA is synthesized in vivo by the method of constructing siRNA expression vector because the siRNA is not easy to operate and the duration of the gene interference is short .

At the same time , the recombinant vector fused to express the green fluorescent protein ( GFP ) gene and the partial rep gene was constructed . siRNA expression vector of the constructed recognition rep gene was co - transfected with the recombinant vector , and the expression of the fusion gene was analyzed by fluorescence microscope and flow cytometry .

II . Anti - SARS virus effect of siRNA in Vero cells

In order to further study the inhibitory effect of siRNA on the replication of SARS virus , Vero cells were transfected into Vero cells by siRNA expression vectors . The inhibitory effect of siRNA on the replication of SARS virus was observed by virus titer assay and indirect immunofluorescence assay .

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【引证文献】