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Establishment of HCV Subgenomic Replicon QSG7701 Cell Line and the CTE-Ribozyme Inhibition of HCV RNA Replication

Author: ChenZhenHua
Tutor: GongGuoZhong
School: Central South University
Course: Internal Medicine
Keywords: Subgenomic replicon Hepatitis C Ribozyme
CLC: R373.21
Type: Master's thesis
Year: 2008
Downloads: 46
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Abstract


The purpose of establishing the RNA transfection HCV Subgenomic Replicon ( HCV subunit replicon) stable transfection and expression of cell lines ; explore CTE- ribozyme of the hepatitis C virus subgenomic group RNA replication . Methods 1. T7 in vitro transcribed RNA kit mediated containing HCVSubgenomic Replicon plasmid pHCV BM4-5 . Converted to RNA . 2 . Establish HCV Subgenomic Replicon stable transfection and expression of cell lines into human hepatoma cell line QSG7701 contain the HCV SubgenomicReplicon of RNA mediated liposome lipofectamine TM 2000 , selected by G418 resistant cloning , expanding culture , the establishment of clonal cell lines transfected . Reverse transcription - polymerase chain reaction (RT-PCR) and Western blot confirmed HCV Subgenomic Replicon RNA replication protein . 3 . Observed ordinary the ribozyme pPHCV5 - R1 the pPHCV5 - R2 and CTE- ribozyme pPHCV5-CR1, pPHCV5-CR2 transiently transfected with HCV subgenomic replicon QSG7701. 48 hours cells were collected after the extraction of total cellular RNA by RT- PCR method to observe the ribozyme cutting effect HCV5'-NCR . Results . Confirmed by RT-PCR and Western Blot , successfully established HCV SubgenomicReplicon RNA QSG7701 stably transfected cell lines . Confirmed by RT-PCR : pPHCV5 - CR1 transfected group no obvious HCV5'-NCR amplified bands , pPHCV5-R1 transfected group visible amplified bands , brightness below untransfected group ; pPHCV5- CR2 visible amplified bands transfected group , brightness below untransfected group , pPHCV5-R2 transfected group visible amplified bands , brightness untransfected close . These results suggest that : CTE - ribozyme the cutting target RNA within the cell efficiency is significantly higher than ordinary ribozyme . Ordinary ribozyme cleavage sites difficult , CTE- ribozyme can be effective cutting ; ordinary ribozyme cleavage sites CTE- ribozyme cutting efficiency is significantly improved . Conclusion 1 . Successfully established HCV Subgenomic Replicon QSG7701 cell lines . 2.CTE- ribozyme within the cell cutting HCVRNA the efficiency significantly higher than ordinary ribozyme . Ordinary ribozyme cleavage sites difficult , CTE- ribozyme also can be performed effectively cutting ; ordinary ribozyme cleavage sites CTE- ribozyme cutting efficiency is significantly improved .

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus) > Enteroviruses and hepatitis virus > Infectious hepatitis
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