人乙酰肝素酶基因启动子驱动的真核细胞表达载体构建、鉴定及功能分析

目的构建人乙酰肝素酶（HPSE）基因启动子驱动的肿瘤细胞特异性表达载体,并分析其活性。方法PCR扩增人类基因组DNAHPSE启动子核心片段,并测序鉴定和分析转录因子结合位点（TFBS）。双酶切插入pEGFP-1的多克隆位点,构建肿瘤细胞表达载体pEGFP-Hp。将经酶切和测序鉴定的重组质粒pEGFP—Hp及质粒pEGFP-1（阴性对照）和pEGFP—N1（阳性对照）分别转染正常人脐静脉内皮细胞（ECV）和不同的肿瘤细胞（肝癌细胞HepG2、喉癌上皮细胞Hep2和慢性白血病K562细胞）。荧光显微镜观察和流式细胞术分析其促转录活性。结果扩增的HPSE启动子核心片段长度为561bp,序列分析与GenBank收录一致,含有3个SPI、4个Ets相关元件、2个早期生长反应基因-1及E47、N—mye和NGFI—p300等TFBS各1个。重组质粒pEGFP—Hp经酶切和测序鉴定与预期结果一致。荧光显微镜观察显示,转染pEGFP-1细胞中均无荧光表达;转染pEGFP—N1细胞均有强荧光表达;转染pEGFP—Hp质粒的ECV几乎无荧光表达,HepG2和Hep2细胞有较强荧光,K562细胞仅有较弱荧光。流式细胞术检测表明,pEGFP—Hp在ECV、HepG2、Hep2和K562细胞的平均转染率分别为3．9％、21．3％、10．8％和6．5％,pEGFP-N1转染率分别为17．1％、24．0％、14．0％和11．0％,二者比值均小于1。结论成功构建了由HPSE核心启动子驱动的真核细胞表达载体,该载体可在肿瘤细胞特异性表达,但其活性需进一步加强。

Construction,identification and functional analysis of eucaryotic cell expression vector modulated by human heparanase gene promoter

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.