| Summary: | 碩士 === 慈濟大學 === 分子生物及細胞生物研究所 === 94 === Severe acute respiratory syndrome (SARS) was an acute respiratory infectious disease that spreaded worldwide in early 2003. The cause was determined as a novel coronavirus, SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. No effective specific treatment has been developed to suppress its infection. Spike protein, a glycoprotein on the viral surface, is crucial for viral attachment and entry into the host cell.
RNA-mediated interference as an antiviral mechanism was originally discovered in plants and then found in other organisms such as Caenorhabditis elegans, Drosophila, and vertebrates. It is an evolutionarily conserved process for the specific suppression of gene expression. Recently, this finding was employed as a technique in anti-virus infections such as human immunodeficiency virus and hepatitis C/B virus. In this study, the RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection.
The purpose of this thesis was to inhibit SARS-CoV spike gene expression of with RNAi strategy. To this purpose, bicistonic vector coding for truncated spike DNA segment (23077-24454) and EGFP was constructed and termed as pIRES2-EGFP-spike. The efficiency of anti-post transcriptional expression of spike gene was determined by measurement of spike mRNA expression and GFP expression in protein levels. Firstly, co-transfection of vector pIRES2-EGFP and each of four shRNA expression vectors into human 293T cell lines was performed and degradation of spike mRNA was determined. However, the inhibition was not significant due to ill transfection efficiency of pIRES2-EGFP-spike vector. So we changed gears and further established HEK 293 cell line, which stably expresses Spike mRNA, then we transfected three designs of shRNA and siRNA respectively into HEK 293 cells. Thereafter, the expression of spike was measured by semi-quantitative RT-PCR and Western blot analysis. The data showed that among the shRNA and siRNA designs of shRNA-HP1206 and siRNA-1 could efficiently inhibit the expression of spike. Our study provides evidence that RNAi could be used as a tool for inhibition of SARS-CoV.
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