Construction of Murine Hepatoma Specific P_(afp)IRES2-EGFP Vector and Its Expression in H22 Cells

Objective To establish a novel minimal AFP promoter operated hepatoma specific expression vector and analysis its specific expression in H22 cells.Methods Genomic DNA from H22 hepatoma cells was used to amplify the minimal AFP promoter through PCR,CMV enhancer was prepared by PCR using pIRES2-EGFP as template.The two DNA fragments were spliced through overlapping PCRto form a chimeric promoting sequence.Purified product digested by Nde I and Nhe Ⅰ was cloned into pIRES2-EGFP to construct the minimal AFP promoter operated murine hepatoma specific PafpIRES2-EGFP expression vector which was verified by PCR screening,restriction enzyme assay(Nde I and Nhe Ⅰ) and DNA sequencing.Then purified PafpIRES2-EGFP plasmid was transfected into H22 hepatoma cells and YAC-1 lymphoma cells through a jetPEI mediated method.Tissue specific expression the recombinant vector was analyzed by fluorescent microscopy.Results A 229bp and a 324bp DNA segments were amplified respectively from genomic DNA of H22 cells and pIRES2-EGFP.Purified PCR products when spliced by SOE PCR which produced a chimeric DNA sequence of 537bp in length as expected.SOE product and pIRES2-EGFP digested with Nde I and Nhe Ⅰ respectively were ligated together by T4 liagase,in the following colony PCR it was showed that we got several positive clones,when further assessed by restriction enzyme assay,they gave rise to a 537bp DNA fragment which was finally proved to be identical to the minimal AFP promoter and ECMV.The recombinant vector were then transfected into H22 hepatoma cells and YAC-1 cells by jetPEI,bright green fluorescence could be observed only on the cell surface of H22 cell rather than YAC-1 cells at 48h after transfection.Conclusion The Authors successfully constructed a murine hepatoma specific expression vector PafpIRES2-EGFP operated by the minimal AFP promoter,the recombinant vector could express exogenous gene effectively in murine hepatoma cells.