[Gene cloning of mIFN-gamma and construction of recombinant adenovirus].

ABSTRACT

OBJECTIVE: To develop a rapid and efficient method for preparing recombinant adenovirus containing mouse IFN-gamma (mIFN-gamma) gene by homologous recombination in E. coli. in order to build a foundation for research into gene therapy of liver fibrosis. METHODS: The target gene mIFN-gamma was amplified by using PCR from the vector pORF5-mIFN-gamma. Once verified, it was cut out by double endonucleases, then connected to the shuttle vector pAdTrack-CMV. The newly constructed vector was linearized by Pme I following transformation to the E. coli. BJ5183, which contained the backbone vector pAdEasy-1. The correct recombinant pAd-mIFN-gamma was selected by endonucleases and by Kanamycin resistance. Again it was linearized with Pac I , then transfected to AD-293 cells by means of Calcium Phosphate method. Finally, the target gene IFN-gamma was identified by PCR and Western blot methods. RESULTS: The target gene mIFN-gamma amplified by PCR was identified by DNA sequencing, which proved that the mIFN-gamma gene consisted of 468 nucleotides and was completely the same with the sequence published on the GenBank. The adenoviral vector constructed by homologous recombination had the gene of interest and the viral could be examined 4-6 days after transfection, and the green fluorescence intensity became greater at about 8-11 days. The adenovirus obtained at the 12th day was digested by protease K and then was amplified by PCR and identified by Western blot. The two methods proved that the adenovirus encoded the target gene mIFN-gamma. CONCLUSION: Preparing recombinant adenovirus containing mIFN-gamma gene by homologous recombination in E. coli. Is a rapid and efficient method. The Ad-mIFN-gamma can be propagated in 293 cell line. It may be used as a novel agent for gene therapy in liver fibrosis.