Construction Of Human Interferon-γ-contained Recombinant Adenovirus

OBJECTIVE: To construct the recombinant adenovirus containing human interferon-γby the method of homogenous recombination in bacteria.METHODS: hIFN-γgene was amplified by polymerase chain reaction(PCR) from plasmid of pORF-hIFNg and subcloned into the plasmid of pGEM-T Easy and the plasmid of pGEM-hIFN-γwas constructed. Then hIFN-γgene was liberated from pGEM-MFN-γand subcloned into pShuttle-CMV vector and turned into transfer plasmid of pShuttle-CMV-hIFN-γ. Adenovirus genomic DNA plasmid of pAdeasy-1 was transformed into BJ5183 bacterial cell and prepared electrocompletent BJ5183 containing pAdeasy-1. pShuttle-CMV-hIFN-γwas linearized with Pme I and transformed into electrocompletent BJ5183 bacterial cell containing pAdeasy-1 and positive clone of homologous recombination was selected. The recombinant adenoviral plasmid pAd-hIFN-γwas obtained by homologous recombination in bacteria. The DNA of identified recombinant plasmid was digested with Pac I and transferred to Ad293 cells to package adenovirus particles.RESULTS: We successfully construct recombinant adenovirus plasmid pAd-hIFN-γand can detect the target gene by PCR technique in exact size. The recombinant adenovirus containing hIFN-γgene was also successfully packeged by transferring Ad293 cells and the cytopathic effect(CPE) was observed. CONCLUSION : Recombinant adenovirus Ad-hIFN-γcan be efficiently and conveniently constructed by homogenous recombination in bacteria ,and which can be used in further study of gene therapy.