论文部分内容阅读
LeY是一种双岩藻糖化寡糖,在大多数上皮来源的肿瘤细胞(包括乳腺癌、卵巢癌等)中高表达.岩藻糖基转移酶Ⅳ(fucosyltransferaseⅣ,FUT4)是合成LeY的关键酶.前期工作发现,FUT4通过增加LeY糖的合成来促进细胞的增殖.但有关FUT4的转录调控机制尚不清楚.本文通过对人FUT4基因近端启动子进行生物信息学分析,并构建不同长度启动子序列荧光虫荧光素酶报告基因表达载体,分析其转录活性.使用First EF程序分析并获得FUT4近端启动子序列,采用PCR法扩增FUT4基因近端不同长度的启动子序列,定向克隆,获得不同长度的启动子重组质粒.重组质粒经双酶切及测序鉴定正确.荧光素酶活性分析不同长度的FUT4基因启动子片段的转录活性.结果显示,pGL6-FUT4-1.2 kb在MCF-7和MDA-MB-231细胞中转录活性明显升高(P<0.05).说明FUT4基因启动子区域定位于转录起始位点上游的-800～-1 600 bp的区域内.
LeY is a bi-fucosylated oligosaccharide that is overexpressed in most epithelial tumor cells (including breast, ovarian, etc.) Fucosyltransferase Ⅳ (FUT4) is a key enzyme in the synthesis of LeY It was found that FUT4 promotes cell proliferation by increasing the synthesis of LeY.However, the mechanism of transcriptional regulation of FUT4 is not clear.In this paper, bioinformatics analysis of the proximal promoter of human FUT4 gene and construction of promoter sequences of different lengths The luciferase reporter gene expression vector was used to analyze its transcriptional activity.First EF program was used to analyze and obtain the FUT4 proximal promoter sequence.The PCR products were amplified by PCR from different lengths of the FUT4 Length of the promoter recombinant plasmid.The recombinant plasmid was identified by double-enzyme digestion and sequencing.Fluorescent activity of different lengths of FUT4 gene promoter fragment analysis of transcriptional activity.The results showed that pGL6-FUT4-1.2 kb in MCF-7 and MDA -MB-231 cells (P <0.05), indicating that the promoter region of FUT4 gene is located in the region of -800 ~ -1 600 bp upstream of the transcription initiation site.