Development Of A Novel Subgenomic HCV Replicon With Tow Small IRESs And Reporter Gene

The infection of Hepatitis C Virus (HCV) is 1-2%in the world, while in China about 3.2%, which is one of causative agents to acute or chronic liver disease.80%of infected individual are at risk for developing chronic liver disease. In addition to inducing liver cirrhosis, a sigenificant proportion of these infetctions also result in hepatocellucar carcinoma. Consequently, HCV-induced chronic liver disease is now recognized as the leading indication for orthotopic liver transplantation in the United States. With the remarkable ability of HCV to estabilish persistent infections that can lead to progressive liver pathology and the poor response of prevalent HCV gemotypes to the current treatment,HCV represents a significant global medical and economic health problem. HCV has been classified as the sole member of the genus Hepacivirus within the Flaviviridae family, identified in 1989 and inducing a trend of study for genetic and protein structure and biochemistry of HCV. HCV have a high level of genetic heterogeneity and thus have been grouped by their degree of sequence identity into six separate genotype and further individed into monerous subtypes. The mechanisms of viral attachment and cellular entry and the precise intracellular steps in HCV RNA replication, virus assembly, and virion release are largely unknown due to the previous lack of suitable cell culture systems for and animal model HCV. To overcome the difficult, numerous exploration praticed, and in 1999, a significant breakthrough occurred when the Bartenschlager laboratory developed the HCV replicon system, a tissue culture system that faithfully mimicked all of the RNA replication on events of the HCV life cycle.Internal Ribosome Entry Site (IRES) are RNA fragment with higher structure, responsible for information of intracellular and virus proteins. Although the internal ribosome entry sites (IRESes) of viral mRNAs are highly structured and comprise several hundred nucleotides, there is a variety of evidence indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. Chappell and his parner analysised the structure of RNA-binding Motif Protein 3 (Rbm3) mRNA and found that a small IRES only 22nt could initiate translation more efficiently about 10 times. With the principle, it is beneficial to moltigenic expression in identical mRNA and construction of a higher efficient HCV subgenomic reporter with resistance and reporter genes.Repoter gene can encode proteins and enzymes that detected quantitatively or qualitatively, that is to say, it is easily to detect its expression product. Fuse the reporter gene and expression or regulation and other purpose gene to form a chimeric gene,then expression under regulation sequence control, eventually, using produce of reporter gene to calibrate purpose gene and get the transformant by fusing with IRES and other purpose gene. Reporter genes, Such as Renilla Luciferase Reporter Gene, Firefly Luciferase Reporter Gene, Beta Lactamase Reporter Gene and Secreted Alkaline Phosphatase Reporter Gene, widely transfected Huh7 cell and detected reporter RNA replication and expression. Renilla luciferase reporter system is the most common and precise one. The activity of renilla luciferase has a close correlation with repoter RNA replication, suggested it is a stable replication mark. Tnen,we could detect the replication of reporter RNA in different time after transfection by detecting luciferase activity. Many sduties confirmed that IRES after the polioencephalomyelitis enhanced replication, activity of reporter gene and other reappearance results than HCV IRES.The replicon system is the most efficient tool to sduty HCV replication. From introduction of definition to identification of adaptive mutation, permissive cell lines, infective of viral genome, screening of antiviral drug, Numerous experts carried out a lot of studys to explort HCV replication mechanism, difiniation function of single protein, identification determinative regulate factors of viral or cell, mechamism of HCV anti the host's antiviral function, detection of interaction in HCV and Huh7 or subseries and screening efficiently antiviral drugs. The development of HCV replicon system has a great practical significant in evaluation antiviral drug, screening drug resistance genes, offering a ideal tool and proposal, which can accelerate development of antiviral drugs. So it is hoping to clean up HCV absolutely.Objective:To construct a novel subgenomic HCV Replicon on the base of pUC19-HCVreplicon, with 2 small IRESs in series, which expresses Renilla Luciferase and Neomycin Photransferase. Construction of a novel HCV subgenomic cell line original of Huh-7 cells with stable expression of Renilla Luciferase. Observation the characterazition of the cell line as a modle in high-through screening anti-virual drugs.Methods:Observation of the expression efficiency of connection in series of IRESs expressing many structure genes. Expression Rnilla Luciferase gene with Neomycin Photransferase expressing at the same time by molecular biology empirical methods. Getting the novel HCV subgenomic replicon cell line after transcription in vitro into replicon RNA, transfection of Huh-7 cells and pressure screening with G418. Then, contrasted cloning efficientcy by inverted microscope in pUC19 HCVreplicon and the novel replicon, detected the efficiency of 22nt small IRES by obserwating the expression of Green Fluorescent Protein (GFP) in Huh-7 cells transcribed plasmids with different IRESs, detected the level of HCV RNA and renilla luciferase in these novel reporter cells by Fluorescent quantitation Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and luciferase assay. Eventually, the novel replicon cells were treated with various concentrations of IFNα2b, RBV for 72 hours and treated with 300IU/ml IFNα2b and lOug/ml RBV for different length of time, at the same time, treated various concentrations of HMG-CoA reducase inhibitors knowed as an HCV inhibitor internationally Simvastatin and Atorvastatin in 72 hours. The last, detected the hRluc in transient transfection system treated with IFNα2b and RBV.Resluts:We observered the expression of GFP in plasmids with different IRES and found that the GFP was no more than EMCV IRES in plasmid with tow 22nt small IRESs connectted in series, although single small IRES more than EMC VIRES. This result suggested the schedule connecting IRESs in series was feasible. Successfully constructed novel HCV subgenomic replicon and cell lines expressed hRluc and Neo. The cloning efficiency of novel replicon was about 2 times more than pUC19 HCVreplicon. The level of HCV RNA in novel subugenomic replicon cells was coincident with expression of hRluc, suggested that Renilla Luciferase was an excellent reporter gene replaced time and ecominic consuming RT-PCR. RBV, Simvastatin and atorvastatin all could inhibit HCV RNA in replicon cells stablely expressing hRluc. the Interferion-α2b was more efficiently than RBV in inhibition HCV RNA in transient transfection system but no effect in stable cells mentioned above.Conclutions:1 The, efficiency of 22nt small IRES was higher than the EMCV IRES, which supported efficiently expression of structure genes.2 Successfully constructed the novel subgenomic replicon with Neo and hRluc, getting the cell lines with stable Rnilla Luciferase expression.3 a lot of experience confirmed that the novel HCV reporter replicon cell line was sensitive to HCV inhibitors and dose dependent with RBV and HMG-CoA reducase inhibitors.4 The novel cell line was not sensitive to IFN-α2b, but the transient transfection system was sensitive.5 Beause of the dependabity with HCV RNA, the Renilla Luciferase could replace tedious and timing, economic consuming RT-PCR to detect anti-viral drugs.The cell line was a new mode for high-through screening.6 The transient transfcecion system was also used for screening anti-HCV drugs.