Abstract:

Objective To construct the tumor cell-specific expression vector modulated by heparanase (HPSE) gene promoter and analyse its activity. Methods The HPSE gene core promoter fragment was amplified by PCR using the total genomic as template, and was identified by sequencing. The transcription factor binding site (TFBS) was analysed. The amplified gene fragment was subsequently cloned into the multiple clone site of pEGFP-1 vector to construct eucaryotic cell expression vector pEGFP-Hp. The vector driven by HPSE core promoter was transfected into human umbilical vein endothelial cell (ECV) and tumor cell lines including hepatoma carcinoma cell line (HepG2), laryngocarcinoma cell line (Hep2) and chronic myelogenous leukemia cell line (K562) through lipofectamine, respectively, and the vectors pEGFP-1 and pEGFP-N1 were used as negative control and positive control, respectively. The activity of reporter gene GFP was detected using fluorescence microscope and flow cytometry after transfection. Results The length of amplified HPSE promoter was 561 bp, and the sequence was accordant with the GeneBank data which included the TFBSs such as 3 SP1, 4 Ets-relevant element, 2 early growth response gene-1, 1 E47, N-myc and NGFI-p300. The enzyme digestion and sequencing identified the constructed vector pEGFP-Hp was consistent with the expectation. Fluorimetric analysis revealed there was no fluorescence expression in all transfected cells of the pEGFP-1 group, and there was hyperfluorescence in pEGFP-N1 group. As for the pEGFP-Hp group, less fluorescence was found in ECV cells, comparatively hyper fluorescence in HepG2 and Hep2 cells, and dim fluorescence in K562 cells. The average transfection efficiencies of pEGFP-Hp in ECV, HepG2, Hep2 and K562 cells were 3.9%, 21.3%, 10.8% and 6.5%，respectively, while those of pEGFP-Nl were 17.1%, 24.0%, 14.0% and 11.0%, respectively, with all the ratios of the two less than 1. Conclusion The eucaryotic cell expression vector modulated by HPSE gene core promoter could be successfully constructed, which could express specifically in tumor cell lines, and its activity should be further enhanced.

Key words: heparanase, promoter, tumor, gene therapy, vector