人乙酰肝素酶基因增强子的初步筛选与鉴定

Preliminary screening and identification of enhancer fragment of human heparanase gene

目的:初步筛选并鉴定出人乙酰肝素酶(HPSE)基因内具有增强子(enhancer,enh)活性的DNA片段。方法:增加Kpn I、Sal I酶切位点,将含有报告基因增强型绿色荧光蛋白(pEGFP)质粒载体pEGFP-N1改造为pEGFP-MU。选取HPSE基因5'端启动子上、下游的DNA序列并分为10个片段,分别命名为enhx(x分别等于1、2、3……10),每段长1 200 bp,相邻两段间重复200 bp。分别扩增上述10个片段和猿猴病毒40(SV40)增强子序列,分别插入pEGFP-MU,以构建重组质粒pEGFP-MU-enhx和pEGFP-MU-SV40e,并用脂质体2000转染肝癌细胞BEL-7402、胃癌细胞SGC-7901,荧光显微镜和流式细胞仪分析上述候选序列对启动子(CMV)转录活性的影响。结果:琼脂糖凝胶电泳和DNA测序结果显示,PCR克隆序列与GeneBank中序列完全一致。酶切电泳和测序结果显示,重组质粒pEGFP-MU-enhx和pEGFP-MU-SV40e构建符合要求。瞬时细胞转染、荧光显微镜和流式细胞仪分析发现,重组质粒pEGFP-MU-enh6、pEGFP-MU-enh7和pEGFPMU-enh8在人BEL-7402、SGC-7901细胞中表达增强,与阳性对照质粒pEGFP-MU-SV40e相似,其余重组质粒表达较弱。结论:成功克隆人HPSE基因10个增强子候选序列片段,并正确构建重组质粒pEGFP-MUenhx;其中第6、7和8候选片段可能具有增强子活性。本研究为后续进一步筛选和鉴定有活性的增强子序列缩小了搜寻范围,并为将来利用HPSE增强子进行肿瘤基因治疗提供了实验依据。

Objective： To screen preliminarily the DNA fragment with enhancer （ enh ） activity in human heparanase （ HPSE） gene.Methods：Plasmid vector pEGFP-N1 was reformed into pEGFP-MU by adding the rest-riction enzyme cutting site Kpn I and Sal I in its DNA sequence .The DNA sequences in the upstream and downstream of human HPSE gene promotor were selected and divided into 10 fragments, named enhx （ x=1,2,3,？10） .Every fragment was 1 200 bp long, and 200 bp was repeated between two adjacent fragments .Above 10 fragments and the enhancer sequence of simian virus 40 （ SV40 ） were amplified and inserted into the plasmid pEGFP-MU to construct the recombinant plasmid pEGFP-MU-enhx and pEGFP-MU-SV40e.The plasmids were then transfected into hepatoma carcinoma cells BEL-7402 and gastric carcinoma cells SGC-7901 using liposomes 2000 .The effects of above candidate sequences on the transcription activity of cytomegalo virus promoter were detected using fluorescence microscope and flow cytometry .Results：Electrophoresis and DNA sequencing demons-trated that cloned target fragments （ enh1-enh10 and SV40 enhancer ） were completely accordant with the sequences registered in GeneBank . The restriction enzyme digestion and sequencing showed that recombinant plasmids pEGFP-MU-enhx and pEGFP-MU-SV40 e fit the bill of experiment .After transient transfection , fluorescence microscope and flow cytometer found that the expressions of recombinant plasmids pEGFP -MU-enh6, pEGFP-MU-enh7 and pEGFP-MU-enh8 in the tumor cells BEL-7402 and SGC-7901 were increased and similar with that of positive control pEGFP-MU-SV40e, and that the expressions of other recombinant plasmids were relatively decreased .Conclusion：The 10 candidate DNA fragments of human HPSE gene enhancer are successfully cloned , and the recombinant plasmids pEGFP-MU-enhx are correctly constructed .The 6-8 th candidate DNA fragments could possess the activity of enhancer .It zooms out the search scope for further screening and identifying active enhancer , and the study provides experimental evidence for future gene therapy using HPSE enhancer .