Study On The Engineered M1GS Ribozymes That Target To The5’ Untranslated Region Of Hepatitis C Virus Genome And Their Intracellular Antisense Activities

According to HCV genome5non-coding regions of the sequence, with the computersoftware simulation, design and build a set for the sequence of different targets in the areaof artificial M1GS ribozyme (M1GS-HCV/C20, andM1GS-HVC/C67,andM1GS-HCV/C76,). By double digestion, PCR and sequencing method confirms thatbuilt the3kinds of M1GS recombinant plasmid cloning success. In addition, the interceptsincluded HCV5’UTR, partial gene fragments, whose success will be inserted to PGEM-9z(f) plasmid M1GS, construction of substrate DNA fragments of different recombinantplasmid-pGEM-HC. Respectively the M1GS of recombinant plasmid gene transcriptionwith pGEM-HC plasmid gene transcription in vitro mix and found M1GS-HCV/C20,andM1GS-HCV/C67and M1GS-HCV/C76activity is quite high. Further in vitroeffectiveness of three M1GS ribozyme gene into reverse contains physical particle pLXSN,construction M1GS eukaryotic expression vector. At the same time, HCV5’UTR DNAfragment inserted to the pGEF-N1plasmid with green fluorescent protein gene upstream,has successfully built under the control of HCV5’UTR fusion expression vector of greenfluorescence. Respectively the M1GS of eukaryotic expression vectors and the greenfluorescence through the fat of total fusion expression vector-transected Huh-7cells (bothexpression of red fluorescent protein plasmid pDsRed2-C1as a Transfection control), theestablishment of an effective M1GS ribozyme intracellular compound screening system.Through changes in fluorescence microscopic observation of green fluorescence and furtherthrough Typhoon9200on the strength of groups of cells in the expression of greenfluorescent scanning. Results found: enzyme M1GS-HCV/C20and M1GS-HCV/C76maysignificantly inhibit the expression of green fluorescent protein, inhibiting respectively77.54±1.97%±1.48%80.35, and enzyme M1GS-HCV/C67Inhibitory effects of green fluorescence is relatively weak, only to58.82±0.88%. In addition, in the experiments forM1GS nuclear reaction between enzyme and substrate in vitro cutting conditions used someexploration.In short, the study of successful construction and screening of three kinds of M1GSenzyme, not only in vitro can be realized on the HCV genome5’UTR, cutting, but in theintracellular targeting also has a clear inhibitory effect of antisense, thereby contributing tonew anti-HCV antisense drugs laid the Foundation for further research and development.