Construction of Recombinant Adenoviral Vector Carrying Mus-?NGF Gene and Its Expression in the MSCs / 听力学及言语疾病杂志

ABSTRACT

Objective To construct the adenoviral vector carrying Mus-?NGF gene and to study the expression of the gene in the marrow mesenchymal stem-cells(MSCs).Methods Mus-?NGF cDNA was cloned from mice glandulae maxillaries whole mRNA by reverse-transcription polymerase-chain-reactions.The Mus-?NGF cDNA was positively cloned into the adenoviral shuttle vector pAdtrack carrying cytomegalovirus promoter(CMV)and tag-protein(GFP),and then homologous recombined into competent BJ5 183 cells with the adenoviral backbone plasmid pAdeasy-1.The recombinant adenoviral plasmid was identified by restriction enzymes and transfected into HEK293 cells to package and amplify recombinant adenoviral particles which was capable of infecting and would express ?NGF and GFP proteins.After the adenovirus infected the MSCs,the expression was observed via fluorescent microscrope and detected using immunocytochemical method.The secretion of NGF was detected by WB.Results The recombinant adenoviral vector carrying Mus-?NGF gene was constructed and confirmed by restriction endonuclease enzyme analysis.The relucent green fluor-light would be observed in the MSCs.IHC showed that Mus-?NGF gene was exactly transcripted and expressed in the MSCs,and the secretion of NGF was confirmed.Conclusion The exogenous gene,such as ?NGF,an be imported into the MSCs efficiently by recombinant adenoviral vector,and can be expressed and secreted successfully in the cells.It improves foundation for the celluar transplant and gene intervention of MSCs expressing NGF in inner ear.