Construction and identification of recombinant adenovirus vector expressing LacZ gene / 医学研究生学报

ABSTRACT

Objective:AIM To obtein a recombinant replication-deficient adenovirus expressing LacZ protein. Methods:The recombinant adenovirus vector engineered to express LacZ gene(Ad-LacZ) was constructed by recombination in E.coli-BJ5183.The replication-deficient adenovirus was then packed in HEK293 cells after transfection with the construct and subsequently identified by X-gal staining,electron microscopic observation and dot blotting.The expression of LacZ in HeLa cells infected by Ad-LacZ could be detected by X-gal stain. Results:Recombinant adenovirus expression vector of LacZ was successfully reconstructed.And the titer of Ad-LacZ virus reached 1.58?10~(9) pfu/ml.Under transmission(electron) microscope,adenoviral inclusions could be found in the nucleus of the infected HEK293 cells. Ad-LacZ virus was comfirmed to be replication-deficient by dot blotting.And the expression of LacZ was observed in the infected HeLa cells.The gene transfer capability of the recombinant virus was correlated with multiplicity of infection and the infection time. Conclusion:Recombinant adenovirus expression vector of LacZ was successfully reconstructed.