本文選題：SARS冠狀病毒 + RNA干擾　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年碩士論文

【摘要】：嚴(yán)重急性呼吸綜合征(Severe Acute Respiratory Syndrome,SARS)是人類在21世紀(jì)初遭遇的烈性傳染病之一,我國(guó)為該病的重要疫區(qū)。其病原體為SARS冠狀病毒(SARS Coronavirus,SARS-CoV)。目前對(duì)其尚無特效的治療方法,因而探索有效的抗SARS病毒新途徑勢(shì)在必行。 RNA干擾(RNA interference,RNAi)是基于RNA水平的抗病毒策略,憑借自身的特異、高效、快速等優(yōu)勢(shì),它已成為抗病毒領(lǐng)域的研究熱點(diǎn)之一。RNAi的發(fā)現(xiàn)和應(yīng)用為治療SARS病毒感染提供了新思路。 本研究擬開展以下兩方面內(nèi)容,觀察siRNA對(duì)SARS病毒復(fù)制的抑制作用。探索抗SARS病毒感染的新途徑。 一.siRNA的設(shè)計(jì)合成及其對(duì)SARS病毒部分復(fù)制酶基因的抑制作用 由于復(fù)制酶基因的表達(dá)是SARS病毒啟動(dòng)轉(zhuǎn)錄的前提,而刺突蛋白在病毒入侵宿主細(xì)胞的過程中發(fā)揮著重要作用,因此選擇SARS病毒復(fù)制酶基因(rep)和刺突蛋白基因(s)的保守序列作為靶基因,設(shè)計(jì)并合成9種siRNA(pS01～09),其中pS01～07針對(duì)rep基因,pS08～09針對(duì)s基因。由于體外合成的siRNA不易操作且基因干涉作用持續(xù)時(shí)間短,故采用構(gòu)建siRNA表達(dá)載體的方法在體內(nèi)合成siRNA。 同時(shí),構(gòu)建融合表達(dá)綠色熒光蛋白(GFP)基因和部分rep基因的重組載體,將已構(gòu)建好的識(shí)別rep基因的siRNA表達(dá)載體與重組載體共轉(zhuǎn)染293T細(xì)胞,通過熒光顯微鏡觀察和流式細(xì)胞儀分析融合基因的表達(dá)情況。結(jié)果表明,針對(duì)SARS病毒rep基因第3504nt-3522nt的siRNA可使rep基因的表達(dá)量下降33%,說明siRNA可特異而有效地抑制基因的表達(dá)。 二.siRNA在Vero細(xì)胞中的抗SARS病毒作用 為進(jìn)一步研究siRNA對(duì)SARS病毒復(fù)制的抑制作用,將構(gòu)建的siRNA表達(dá)載體分別轉(zhuǎn)染Vero細(xì)胞,獲得穩(wěn)定轉(zhuǎn)錄siRNA的克隆細(xì)胞株V/pS01、03、05～09及對(duì)照細(xì)胞株V/pSN,然后以BJ01株病毒懸液感染克隆細(xì)胞,通過病毒滴度測(cè)定和間接免疫熒光實(shí)驗(yàn)觀察siRNA對(duì)SARS病毒復(fù)制的抑制作用。結(jié)果表明,針對(duì)rep基因和s基因的siRNA(pS05和pS08)呈現(xiàn)出了良好的抗病毒效果,在接種SARS病毒后均未檢測(cè)到病毒的特異性抗原,且病毒滴度下降10~3～10~4倍。另外,通過實(shí)時(shí)定量RT-PCR檢測(cè)病毒感染的細(xì)胞上清中rep基因的含量,結(jié)果顯示,細(xì)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 .

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 陳杰斌;RNA干擾技術(shù)對(duì)柯薩奇B組3型病毒的抑制作用研究[D];南華大學(xué);2006年

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本文編號(hào)：1823830