| Severe acute respiratory syndrome (SARS) whose the other name is atypical pneumonia in China is a newly emerged disease caused by a novel coronavirus, and it can lead to panic in the society because of its droplet infection and high mortality. According to the existed reports and researches, WHO declared that a novel coronavirus, SARS-associated coronavirus (SARS-CoV), is associated with the pathogenesis of SARS. Although SARS has been controlled throughout the world, it is very important to find new methods to fight against this disease in order to prevent its return. To date, no effective and specific therapeutic methods can be used to treat patients suffering from SARS-CoV infection. Although glucocorticoid had good effect in clinic, there is a controversy that how to use it can be more reasonable because this drug has great side effects if it is used incorrectly. Meanwhile, the other drug used widely is ribavirin, but researchers didn't find it has antiviral effect for coronavirus. Convalescence serum of patients with SARS can be used to treat the other patients under critical care, but this method was limited because of its high risk. In addition, although combination of traditional Chinese medicine (TCM) and western medicine treatment has some active effects, its mechanism can't be confirmed. In theory, ideal strategy to control SARS is available vaccines. However, due to the time-consuming course of developing novel vaccines, the vaccines targeting SARS-CoV can not be utilized in the near future. Other theoretical strategies to control pathogens may include antibiotics, antisense oligonucleotides, RNA interference (RNAi) and so on, which are all under developing. Among these strategies, RNAi may represent one perspective potential candidate to overcome SARS-CoV infection. RNAi is a phenomenon in which small double-stranded RNA molecules induce sequence-specific degradation of homologous single-stranded mRNA. It can be induced This work is supported by National Key Basic Research Development Plan of China("973" Projects): Fund for the Basic Research of SARS Prevention(2003CB514108). through transfection or microinjection of long double-stranded RNA in plants and invertebrates. The double-stranded RNA is cleaved into 19- to 23-nt RNA fragments known as small interfering RNAs (siRNAs) by Dicer. siRNAs are incorporated into a ribonuclease enzyme complex known as the RNA-induced silencing complex (RISC). The antisense strand of siRNA within the RISC serves as a guide for sequence-specific degradation of homologous messenger RNAs. To date, with the rapid development of technology, RNAi is not only a powerful tool for studying genes function, but also a potential and ideal gene therapy strategy for treatment human disease. Beside RNAi has been used to suppress tumor growth, it also has been used to inhibit some viruses replication and viral proteins expression, such as HIV, HCV, HBV and influenza virus. According to the above researches, inhibition SARS-CoV replication and infection in the present study has theoretical background. Therefore, the hairpin siRNA template oligonucleotides targeting Leader and Nsp4 genes of SARS-CoV were designed and synthesized according to the whole genomic sequence of SARS-CoV and the manual of pSilencer 3.1-H1 vector. The products of annealing the hairpin siRNA template oligonucleotides respectively were cloned into vector pSilencer 3.1-H1 to construct the 4 kinds of plasmids expressing siRNA. At the same time, Leader and Nsp4 genes of SARS-CoV were obtained by PCR amplification. When the two genes were cloned into the pEGFP-N1 vector, two recombinant plasmids which express Leader+EGFP and Nsp4+EGFP fusion proteins respectively were obtained. After co-transfection, RNAi effects that target the targeted genes, induced by the 4 kinds of plasmids expressing siRNA, were confirmed through FACS and RT-PCR in HeLa cells. Finally, CPE, MTT array and plaque reduction array were finished in P3 laboratory to determine final effects of RNAi targeting SARS-CoV. Here, we rep...
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