Construction and identification of recombinant adenovirus vector containing MDR1-CD∷upp gene driven by human MDR1 promoter

Objective To construct a recombinant adenovirus vector containing human mdr1-CD∷upp gene which regulated by mdr1 promoter.Methods The promoter of the human mdr1 gene amplified from human genomic DNA by PCR was inserted into pGEM-T and PGL3-enhancer vector respectively.CD∷upp gene derived from the plasmid PORF-CD∷upp was inserted into the backward position of mdr1 promoter in the PGL3-enhancer vector.Subsequently,the obtained mdr1-CD∷upp gene was subcloned into an adenovirus shuttle plasmid pAdTrack.After being identified by endonuclease,PCR and sequencing,the recombinant shuttle plasmid pAdTrack-mdr1-CD∷upp was co-transformed into BJ5183 cells with the adenovirus backbone plasmid pAdeasy-1 to obtain the homologous recombination.The adenovirus was generated in 293 cells.Methods such as PCR and fluorescence microscopy were employed to identify the generated recombinant adenovirus.Results Recombinant mdr1-CD∷upp adenovirus were constructed successfully and the titer of virus was 3.0×10~(10) pfu/ml.Conclusion The recombinant adenovirus Ad-mdr1-CD∷upp might be an important base of the further research on its target gene therapy of multidrug resistance in cancer.