Construction and identification of recombinant adenovirus vector encoding ?-galactosidase with modified AdEasy system / 第三军医大学学报

ABSTRACT

Objective To construct a recombinant adenovirus encoding ?-galactosidase gene(?-gal),which may be used to treat the lactose intolerance caused by lactase deficiency,with modified AdEasy system.Methods The sequence fragment of ?-gal was cloned into the shuttle plasmid pAdTrack-CMV,and the homo-logous recombination was completated with backbone plasmid pAdEasy1 in the E.coli BJ5183 to construct the recombinant adenoviral plasmid.The adenoviruses were packaged and amplified in the HEK293 cells mediated by liposome.The viral titre was measured by limiting dilution assay and the transfection efficiency was observed by X-gal histochemical staining.Results The recombinant adenovirus with ?-gal was constructed successfully and the viral titre was(2.5~4.0)?1011EFU/ml.More than 70% HEK293 cells were transfected when the MOI was 10.Moreover,more than 65% cells were blue after X-gal histochemical staining.Conclusion Recombinant adenovirus carrying ?-gal with high titre and efficient transfection is constructed successfully by AdEasy system,which supplies valuable experimental basis for the gene therapy to the lactose intolerance caused by lactase deficiency.