Construction and Identification of Recombinant Adenovirus Containing Human p27 Gene

Objective:To construct and identify the recombinant adenovirus plasmid containing exogenous p27 gene and EGFP gene and to detect the gene expression.Methods: Genetic engineering techniques,including plasmid extraction,agarose gel electrophoresis,restriction enzymolysis,connection,transformation of competent cells,were used to construct the recombinant plasmid of pCMV-IRES2-EGFP-p27.The adenovirus plasmid pAdeno-CMV-p27-EGFP was constructed by adeno-x expression system.The plasmids were identified by enzymolysis and PCR.Recombinant adenovirus vector was packaged by cell line 293.The expression of EGFP was detected under the fluorescence microscopy.Results: pCMV-IRES2-EGFP-p27 was successfully constructed and identified by enzymolysis and sequencing.Fluorescence microscopy revealed that the expression of EGFP could be detected at 48th h after the plasmid was transfected into astrocytes,and the expression of EGFP was increased to the peak at 48-72 h.The adenovirus plasmids pAdeno-CMV-p27-EGFP was successfully constructed and identified by enzymolysis and PCR.The CPE and the expression of EGFP could be observed after the plasmid pAdeno-CMV-p27-EGFP was transfected.Conclusion: The adenovirus plasmid containing human p27 gene was constructed successfully by adeno-X system.It will establish a foundation for the future research about the relationship of chronic graft dysfunction and immunological tolerance.