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Construction and identification of recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant
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Abstract:
Objective: To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant , laying a foundation for gene therapy research of malignant tumors. Methods: The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus; the products were cotransfered into HEK-293 cell line with helper plasmid PJM17. The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results:The -p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells (1×1011pfu/ml). The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR. Ad. NT4p53Ant had strong killing effect on HepG2 cells. Compared with Ad.GFP, Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells. Conclusion: The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques，laying a foundation for further research of gene therapy of cancer.
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