Study On The Targeted Cleavage Activity Of M1GS Ribozymes Specific For The Core Gene Of HCV

Hepatitis C virus (HCV) is one of important viruses threatening to human health. At present, there is no specific, effective treatment measures, it is important to exploring a new type of anti-HCV treatment strategy.In this study,we chose the core gene (C) of HCV genome as a target gene because it is relatively conservative, and plays a key role in the process of viral replication. Aimed at our potential sites (No.52, 167, 347, 438) in the mRNA of the target gene, and based on the catalytic subunit(M1 RNA) of E. coli RNase P, we have constructed four corresponding sequence-specific M1GS ribozymes.Through the extracellular test, we have studied their targeted cleavage activity of the four M1GS ribozymes. The results showed that only the M1GS ribyzyme aimed to the 167th site could produced the targeted cleavage, and led to two products, one is 167nt in length, another is 420nt in length. Nevertheless,the targeted cleavage activity of other three M1GS ribozymes were not obvious.To further determine if the M1GS ribozyme has an introcellular inhibition to the expression of target gene or not, we firstly construced two recombinant plasmids which could express in eukaryotic cell, one was the pcDNA-M1GS2, the other was the pcDNA-HCV/core, and then co-transfected the two eukaryotic expressive plasmids into HeLa cells. By semi-quantative RT-PCR and western blot, we found that the M1GS ribozyme aimed to the 167th site could not only significantly lower the level of the target mRNA, but also could greatly reduce the expression of HCV core protein.In short, through the study of this project, we have successfully got a potential M1GS ribozyme which possessed extracelluar and introcellular cleavage activities to the target mRNA of HCV C gene, so as to provide direct experimental materials for the future research(such as the anti-HCV infection in host cells, and anti-HCV infection in vivo, etc.), as well as laid a solid foundation for the anti-HCV treatment research using the new ribozyme technology—M1GS.