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


CONCLUSIONS
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.
Xue
Hong-Li
HL
Department of Hematology & Research Lab of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China.
Lin
Shi-Jia
SJ
He
Dong
D
Meng
Wen-Tong
WT
Niu
Ting
T
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D008103:Liver Cirrhosis / Q000628:therapy; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D007371:Interferon-gamma