[Construction of a hepatoma-targeting vector of adeno-associated virus containing human alpha-fetoprotein promoter and wild p53 gene in gene therapy of liver cancer]

Abstract

Objective: To construct plasmids that express target genes in hepatoma cell line using adeno-associated virus (AAV) vectors containing human AFP promoter.

Methods: Primers containing specific enzyme-cutting sites were designed to amplify the alpha-fetoprotein promoter (AFP promoter) from human genome. The promoter was cloned into pTR-UF5, a plasmid containing GFP reporter gene, resulting in the recombinant AAV plasmid containing the reporter gene (rAAV-AFP-GFP). Blunted ligation was used to construct the recombinant AAV vector plasmid containing human wild p53 gene (rAAV-AFP-53). The plasmid rAAV-AFP-GFP was used to transfect the AFP-expressing Hep G(2) and non-AFP-expressing 293 cell lines, respectively, to measure the function of the cloned AFP promoter. Flow cytometry was used to measure the effect of rAAV-AFP-53 on hepatoma cell line HLE.

Results: rAAV-AFP-53 and rAAV-AFP-GFP were verified by DNA sequencing and enzyme digestion to carry human AFP promoter. Cell transfection of rAAV-AFP-GFP showed selective expression in AFP-positive hepatoma cell lines with a transfection rate of 36.5%; rAAV-AFP-53 induced apoptosis rate was 73.88%.

Conclusion: Two adeno-associated virus plasmids are successfully constructed that carry p53 gene and reporter gene, respectively, guided by AFP promoter. The former one shows a hepatoma-specific apoptosis-inducing effect.