Establishment Of A Mouse Model With Long-term Expressing HCV Core Protein

Viral hepatitis C is a major public health concern worldwide. HCV causes chronic hepatitis, cirrhosis and liver cancer. Approximately 170 million people suffer from this infectious disease around the world. Today no therapy is proved to be enough valuable to hepatitis C treatment. IFN-αand ribavirin combination therapy is the standard clinical treatment, but this treatment is only less than 50% of patients.HCV is an enveloped, positive-sense RNA virus of the family Flaviviridae. The 9.6-kb genome encodes one large polyprotein that is processed by viral and cellular proteinases to produce the virion structural proteins (core protein,glycoproteins E1,E2 and P7) as well as nonstructural proteins (NS2 through NS5B). The core protein which is derived from the N-terminus of the viral polyprotein forms the viral nucleocapsid. The amino acid sequence of this protein is well conserved among different HCV strains in comparison with other HCV proteins. More and more reports indicate that core protein is related with carcinogenesis of HCV. However, HCV has host specificity. The mechanism and function of HCV and core protein were mostly studied in cell models. Lacking of appropriate small animal models that can infect HCV hampers the study at the whole level in animal.In china, the HCV strain is mainly genotype 1b. And the patients infected with HCV-1b is less effective using IFN-α. So study of HCV-1b core protein in HCV persistent infection and HCC pathopoiesis mechanism has important significance for HCV prevention and cure. We established a mouse model which stably express HCV 1b core protein. And the model could be used for evaluating the effect of shRNAs which target HCV core gene. The main research content and results are as follows:1. We constructed an eukaryotic expression vector pGL3-attB-Core-Fluc. This vector contains HCV core protein, report gene Fluc and attB site that can be recognized by phage integrase. To investigate whether these constructs could express core protein and Fluc, pGL3-attB-Core, pGL3-attB-Fluc and pGL3-attB-Core-Fluc were transfected into Huh7 cells respectively. The expression level of core protein in cells transfected with pGL3-attB-Core-Fluc was equivalent of that in cells transfected with pGL3-attB-Core. But the expression level of Fluc was lower than that in cells transfected with pGL3-attB-Fluc. The same result was also disovered in mice.2. By site-special recombination of the phage ?C31 integrase,we established a mouse model with stable expression of HCV core protein. pGL3-attB-Core-Fluc was injected with the integrase expression vector pCMV-Int into the liver of mice by hydrodynamics-based procedure. The experiment confirmed that when pCMV-Int was included, core protein and Fluc integrated to mpsL1 site located at mice chromosomal 2 and got a stable expression.3. Based on the stable mouse model, the shRNAs targeted the highly conserved core protein region of the HCV genome were evaluated in vivo.Based on pSilencerTM2.1-U6 neo vector, we constructed shRNAs expressing plasmids: shRNA452, shRNA479 and shRNA523. In cultured cells, the results revealed that the luciferase activities and core protein expression were reduced about 40-55% in the cells co-transfected with shRNA452, shRNA479 and shRNA523. The inhibitory effects of these three shRNAs had no statistical difference. In the transient mouse model, the effect of shRNA-523 was detectable as early as 24 hr and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48hr post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6hr and 12 hr after injection respectively, and the inhibitory effect persisted for 1 day after a single injection.In conclusion, this study combined hydrodynamic transfection and phage integrase system to establish a mouse model which can stably express HCV core protein. Moreover, shRNA targeting HCV core protein can effectively downregulate core gene and reporter gene expression in the liver of mouse model. This luminescence-based method allows continuous monitoring of the kinetics of HCV core protein inhibitors in live animal. This novel and simple method can be used for screening anti-HCV compounds.