0bjective To observe the
cell membrane penetration of
protein transduction domain (PTD)-
HBeAg fusion
protein in vitro.
Methods The sequence of
trans-activator of transcription (Tat)-PTD was synthesized and the whole
HBcAg gene was amplified by
polymerase chain reaction (
PCR).Overlap extension
PCR was employed to fuse Tat-PTD and whole
HBcAg gene.Then the fusion
gene was cloned into prokaryotic expression vector pMAL-c2X.The correct vector was transformed into E.coli Rosetta-gamiTM 2(DE3),and the
protein was induced by isopropyl β-D-1-thiogalactopyranoside(
IPTG).
Western blot was used tO identify the
protein. Furthermore,the fusion
protein PTD-
HBcAg was purified by
affinity chromatography.
HBcAg protein expressed using the same
methods was employed as eontr0l.The purified
protein was added tO HuH-7
cell culture,then the transduction of PTD-
HBcAg and
HBcAg in
cells were detected by
indirect immunofluorescence assay (IFA).Results The fusion
protein was effectively expressed in E. Coli and purified by
affinity chromatography.Both purified PTD-
HBcAg and
HBcAg could be recognized by
HBeAg monoclonal antibody in
Western blot analysis.IFA visualization showed that PTD-
HBeAg could be introduced into HUH-7 ceils while
HBcAg only could not be detected in
cells.Conclusions PTD-
HBcAg fusion
protein can be expressed effectively and purified in prokaryotic expression system.PTD could mediate
HBcAg penetrating eell
membrane into the
cells.