Study On The HCV Ns5b Gene Expression Disruption By RNAi

Hepatitis C virus (HCV), the etiologic agent responsible for non-A, non-B hepatitis in humans, is a leading cause of chronic liver diseases. About 75% of chronic HCV infections can proceed into the development of liver cirrhosis, among these 25-30% developed into hepatocellular carcinoma and liver failure .Over 170 million people worldwide are estimated as being infected by HCV, the infected numbers of our country account for 5 million . HCV infection and its related diseases has been viewed as a growing threat to human health worldwide and the severe problem of social public health. Currently there were no specific prophylactic and therapy.HCV are a group of single-stranded RNA viruses that belong to the Flaviviridae family and hepavirus .All HCVs possess a positive-sense RNA genome of approximately 9600 nucleotides . The HCV genomic RNA contains a single open reading frame and the non-code region of both laterals related to the replication and translation. HCV NS5B protein is encoded by ns5b gene, an RNA -dependent RNA polymerase that is responsible for the viral RNA synthesis.Due to no corresponding enzymes in the host cell and the importance of NS5B proteinin the replication ,NS5B protein has been regarded as an ideal target of anti-hcv infection.RNAi (RNA interference) is a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA . Since Fire et.al. found the phenomenon observed in the nematode caenorhabditis elegans for the first time in 1998 , consequently similar processes have been described for Drosophila melanogaster,trypanosome , mammals including humans.The mechanism is that siRNAs is the mediator,which can induce the RISC to the target mRNA and degrade it .Recently there was great progress in the specific gene therapy and anti-virus,and RNAi has been a focus of RNA molecular therapy.This study we acquired the coding region of HCV ns5b gene by PCR of HCV full length genome and construct the recombinant plasmid pEGEP N3-ns5b ;With the different concentration of G418 in the culture medium,we think the selection concentration of G418 for HepG2 cell is 800μg/ml ;The recombinant plasmid was transfected into HepG2 cell by LipofectAMINE2000 cells containing stable transformants were selected by the ability of resistance to G418 and isolated with the limited dilution. The stably transfected cell line expressing NS5B-EGFP fusion protein was obtained by the detection under fluorescence microscope and RT-PCR. According to the gene sequence and secondary structure of HCV ns5b,we design the siRNAs targeting ns5b gene following with the requirement for siRNAs design from Tuschl et.al and synthesize it from Dharmacon company ; HepG2 cell stably expressing NS5B-EGFP protein was trasfected by synthesized siRNAs with electroportion , the non- transfected cell and non-specific siRNAs transfected cell are consideredas control group ;Inhibitory effect of siRNAs was investigated by fluorescence microscope with DAPI dyeing and by semi-quantitative RT-PCR. linhibition of HCV ns5b gene expression by synthesized siRNAs provide the new method of anti-HCV study .