Abstract
Two approaches have been developed to construct plasmids that mediate RNA interference to inhibit the replication and expression of HBV in 2.2.15 cell. The overlapping PCR extension and restriction enzyme-digestion were used to generate DNA fragments encoding designed shRNA based on sequences of ORF C of HBV genome. The pU6 derived vectors were constructed to develop plasmid based shRNA delivery systems termed pU6/HBVi. There were significant reductions in the expression of HBsAg and HBeAg between cells transfected with pU6/HBVi and control groups (as to HBsAg: P < 0. 01; and HBeAg: P < 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 × 107 to <5 × 102 copies with or without pU6/HBVi. These results suggested that shRNA delivery by recombinant plasmids harboring shRNA encoding DNA fragment of interest generated either by overlapping PCR extension or restriction enzyme-digestion, could inhibit expressions of viral proteins and reduce viral replications. The pU6 derived plasmids might be a useful shRNA delivery system in mammalian cells. In addition, we found siRNA based on stealth 2311 was a potent RNAi target of HBV genome.
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Acknowledgement
We are grateful to Dr. Hongwei Liu (Department of Plastic Surgery, the First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province 510630, P.R. China) for help in revising the manuscript.
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Zhang, HB., Wu, J., Xian, J. et al. Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro. Mol Biol Rep 35, 465–472 (2008). https://doi.org/10.1007/s11033-007-9108-0
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DOI: https://doi.org/10.1007/s11033-007-9108-0