[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].
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%.


CONCLUSIONS
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.
Cheng
J
J
Department of Surgery, People's Hospital, Beijng Medical University, Beijing 100044, China.
Leng
X
X
Peng
J
J
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D018390:Gene Targeting; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy*; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D000509:alpha-Fetoproteins / Q000235:genetics*
D008164:Luminescent Proteins; D016159:Tumor Suppressor Protein p53; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins