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The hepatitis C virus (Hepatitis C Virus, HCV) , hepatitis C pathogenesis of the body , there are around 170 million hepatitis C infection . HCV infection may have a variety of different clinical manifestations , and the majority of infected people develop chronic hepatitis , and thus more likely to develop liver cirrhosis and hepatocellular carcinoma . Because of the serious harm of the hepatitis C , in recent years , the study of the hepatitis C virus , especially virus molecular biology research progress is very rapid . Lohmann in 1999 first reported on the Science HCV RNA replication in vitro model of selective bicistronic subgenomic group HCV RNA replication subsystem , replicon in the human hepatoma cell line Huh7 can be high levels of self- replication , which is recognized breakthrough milestone . Copy subsystem established, having a significance of HCV pathogenesis and treatment of drug screening and vaccine research . This work is intended to construct a can intuitively rapid reaction cells the number of HCV RNA recombinant HCV 1b replicon , without taking advantage of HCV RNA by RT-PCR to detect intracellular after G418 selection , for the qualitative evaluation of some compounds for HCV resistance has important significance. Construction of the recombinant vector of the basic ideas : the use of AscI and PmeI restriction enzyme sites will HCV 1b subgenomic group replication within the promoter of the G418 resistance gene neo fragment , cut , while using the PCR method to obtain added the above two restriction sites at the end EGFP fragment , obtained by in vitro ligation excluding a G418 resistance gene , and the genomic clone containing the EGFP reporter gene recombinant HCV1b subunit pEGFP -1b ; the cDNA clones obtained after in vitro transcription, recombinant HCV 1b subunit genomic RNA application of liposomal transfection technology transiently transfected Huh7 cells , but not detected as expected the results to the cell fluorescence . However , work in the same experimental conditions using the pEGFP-N1 plasmid was transiently transfected Huh7 cells , can be clearly observed cell fluorescence .
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