Objectives To construct the recombinant adeno-associated virus serotype 9 containing ribozyme gene (R65) targeting nuclear factor-κ B (NF-κB), and investigate the inhibitory effect of rAAV9-eGFP-R65 on activation of NF-κB as well as on protein expression of NF-κB P65 in HeLa cells.
Methods The synthesised ribozyme gene targeting NF-κB was inserted into the plasmid pFB-CMV-eGFP with definite direction, and packaged into the recombinant adeno-associated virus serotype 9 by three plasmids co-transfection, then recombinant adeno-associated virus was purified by cesium choride density centrifugation. The purity of recombinant virus rAAV9-eGFP-R65 was observed and verified by transmission electron microscopy and SDS-PAGE and viral tite was checked by GFP. Finally, HeLa cells were infected by the recombinant adeno-associated virus, the activation of NF-κB and protein expression of P65 were analysed by electrophoretic mobility shift assay  and Western blot.
Results The high expression of green fluorescence protein expression in HEK293 and HeLa cell lines were found under fluorescent microscope. Electron microscopy and SDS-PAGE test indicated that recombinant adeno-associated virus rAAV9-eGFP-R65 was successfully constructed, and the titre of the virus reached 4.63×1012 vg/ml. Western blot and EMSA showed that the protein expression of P65 and the activation of NF-κB in HeLa cells were markedly inhibited after transfection with rAAV9-eGFP-R65.
Conclusions Activation of NF-κB and expression of NF-κB p65 protein in HeLa cells are effectively inhibited by recombinant adeno-associated virus rAAV9-eGFP-R65, which lays the foundation for further researches into therapy nuclear factor-κ B related ischemic diseases.
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