Construction and expression in vitro of a mifepristone inducible recombinant adenovirus carrying enhanced green fluorecence protein / 医学研究生学报

ABSTRACT

Objective To construct a mifepristone inducible recombinant adenovirus carrying enhanced green fluorecence protein for future gene therapy in vivo.Methods Full length red fluorescent gene and mifepristone regulator cassette was subcloned into PDC313 shuttle plasmid.The positive clone was identified by sequencing.Recombinant adenovirus were produced after cotransfection of the shutter vector PDC-EGFP and adenovirus DNA helper plasmid pBHGloxE1,3Cre into 293 cells.The recombined adenovirus was purified by CsCl density gradient centrifugation and its titer was determined by end point dilution assay.Fluorescence microscopy and FCM were confirmed by the activation of this regulatable recombinant adenovirus vector in infected cells.Results The adenovirus vector containing enhanced green fluorecence protein gene was identify by PCR.The virus titer of Ad-RUEGFP was 6.6?1010pfu/ml.Without the absence of mifepristone,no significant enhanced green fluorecence protein activation was observed,whereas in the presence of mifepristone activation of the enhanced green fluorecence protein was positive correlation with the inducer under definite range.Conclusion A replication-defective recombinant adenovirus vector containing enhanced green fluorecence protein was successfully constructed,which can be inducible by mifepristone.