克隆猿猴病毒40增强子修饰人乙酰肝素酶基因核心启动子及活性分析

摘要

目的:克隆猿猴病毒40(simian virus 40,SV40)增强子序列,修饰人乙酰肝素酶(heparanase,HPSE)基因核心启动子,并分析其活性.方法:PCR扩增SV40增强子序列并测序鉴定,酶切连接插入到pEGFP-HPSEp中指定克隆位点构建重组质粒pEGFP-HPSEp-SV40enh.将原质粒、重组质粒和阳性对照质粒分别转染肝癌细胞(HepG2)、喉癌上皮细胞(Hep2)和慢性白血病细胞(K562).荧光显微镜观察和流式细胞术分析各质粒促转录活性.结果:扩增的SV40增强子序列长度为237 bp,序列分析与GeneBank收录一致.重组质粒pEGFP-HPSEp-SV40enh经酶切和测序鉴定与预期结果一致,SV40增强子序列插入到指定克隆位点.荧光显微镜观察显示,转染pEGFP-HPSEp-SV40enh的HepG2、Hep2和K562细胞均有较强荧光表达;转染pEGFP-HPSEp的细胞荧光强度均较弱.流式细胞术检测表明,pEGFP-HPSEp-SV40enh在HepG2、Hep2和K562细胞的平均转染率分别为3.7%、6.0%和5.5%,pEGFP-HPSEp转染率分别为4.8%、13.4%和7.7%,两者比值均小于1.结论:成功克隆了SV40增强子序列并构建了重组质粒pEGFP-HPSEp-SV40enh,SV40增强子可以初步提高HPSE启动子的活性.%Objective:To modify heparanase gene core promoter with amplified SV40 enhancer DNA sequence and analyze its activity. Methods:SV40 enhancer sequence was amplified and inserted into the multiple clone site of pEGFP-HPSEp to construct a recombinant plasmids named pEGFP-HPSEp-SV40enh. The vector driven by HPSEp- SV40enh and HPSEp were transfected into human tumor cell lines including hepatoma carcinoma cell line (HepG2) , laryngocarcinoma cell line(Hep2) and chronic myelogenous leukemia cell line(K562) , respectively. The activity of reporter gene GFP was detected using fluorescence microscope and flow cytometry after transfection. Results:The length of amplified SV40 enhancer was 237 bp and the sequence was accordant with the GeneBank data. The enzyme result of digestion and sequencing of the constructed vector pEGFP-HPSEp-SV40enh was consistent with the expectation, SV40 enhancer sequence was inserted into the allocated multiple clone site. Fluorimetric analysis revealed that hyperfluorescence was found in pEGFP-HPSEp-SV40enh group and less fluorescence was found in pEGFP-HPSEp group,The average transfection efficiencies of GFP-HPSEp-SV40enh in HepG2, Hep2 and K562 cells were 3. 1% , 6. 0% and 5. 5% , respectively, while those of pEGFP-HPSEp were 4. 8% , 13.4% and 1.1% , respectively, with all the ratios of them less than 1. Conclusions: SV40 enhancer sequence is amplified successfully and pEGFP-HPSEp-SV40enh could be successfully constructed, SV40 enhancer might improve the activity of HPSE promoter.