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

Modifying heparanase core promoter with SV40 enhancer

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 3.7%,6.0% and 5.5%,respectively,while those of pEGFP-HPSEp were 4.8%,13.4% and 7.7%,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.