Study On The RNA Interference Of RdRp Gene Of SARS Coronavirus

Severe Acute Respiratory Syndrome (SARS) is a highly contagious disease with high mortality, and there is no effective remedy to treat it. Viruses isolated from SARS patients have been confirmed to be a new type of coronavirus. RNA interference (RNAi) can cause degradation of an mRNA homologous in sequence to the double-stranded RNA (dsRNA). This makes it to be a powerful tool in determining gene function, as well as a specific approach in gene therapy. Small interfering RNAs (siRNAs) with high inhibitory efficiency against target gene have been developing into specific medicaments to treat virosis, cancer and hereditary diseases. In this project, we focused on the screening of effective siRNAs against the key gene of SARS coronavirus, for the further investigations with therapeutic purpose.Sequence analysis showed that RNA dependent RNA polymerase (RdRp) gene is the most conserved gene in the virus genomes. RdRp, which is the pivotal to turn on the life cycle of the virus in host cell, is the most important protein of SARS coronavirus. Therefore, RdRp gene was selected as the target in our study. A safe, efficient and reliable method was first designed to acquire the RdRp gene. According to the principle of DNA-shuffling and the sequence of RdRp gene, oligos were synthesized with complementary ends to primer each other during the PCR process to assemble the full-length gene. The assembled RdRp gene had the exact sequence as expected.Three kinds of mammalian expression plasmids were then constructed, using EGFP (enhanced green fluorescent protein) as gene marker to facilitate the consecutive...