Construction and identification of small hairpin RNA vector targeting HBV core protein in HepG2.2.15 cells

Abstract

Objective To construct eukaryotic expression vector of small hairpin RNA vector targeting hepatitis B virus core protein in HepG2.2.15 cells,and to verify the interfering effect.Methods To design and synthesize small interfering RNA sequence against HBc gene (ayw subtype) which locates between 2147 bp to 2165 bp in the HBV genome.The siRNA fragment were inserted into pSilencer3.1-H1 neo vector.The recombinants were transformed into E.coli DHSα successfully.The recombinant plasmids were identified by Eco RI enzyme digestion and sequenced.Finally,The HepG2.2.15 cells were transfected with the vector by using lipofectamine method to identify its interfering effect.Results The eukaryotic expression vector containing small hairpin RNA targeting HBV core protein was constructed successfully and named as pshRNA-HBc.The recombinant plasmids were identified by SalI or EcoRI enzyme digestion and sequenced.The silencing effect was detected by using revere transcription polymerase chain reaction (RTPCR) and the secretion of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells.The result showed that the interfering effect was specific and efficient.Conclusion The small interfering RNA targeting HBV core protein in HepG2.2.15 cells was constructed successfully,which may be useful for studying the biological functions of HBc in the future. Key words: Hepatitis B virus; Viral core proteins; RNA