Preparation Of Sirna And Its Application In The Inhibition Of Hepatitis B Virus Replication In E. Coli Fermentation

RNA interference, the process of specific mRNA degradation induced by homologous dsRNA, has been widely used for target-specific gene silencing, and holds great therapeutic potential in clinical applications, especially in the field of anti-cancer and anti-virus therapy. Yet one major challenge is the large-scale production of siRNAs in low cost. We described in this paper a cost-effective method to prepare siRNAs based on cloning, fermentation, digestion and purification(CFDP). siRNAs prepared with CFDP specifically and dramatically inhibited the hepatitis B virus replication both in human liver cancer cells and in mouse livers.In the first chapter, we described the construction of an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli. The fermentation condition was optimized to obtain high expression level. dsRNAs were purified by a single step affinity chromatography using CF-11 column.In the second chapter, we described the preparation of recombinant Escherichia...