hMAM启动子／增强子调控表达载体构建和调控作用

目的构建人乳腺珠蛋白（human mammaglobin，hMAM）启动子／增强子调控报告基因表达载体，探讨hMAM启动子／增强子序列在乳腺癌细胞中的特异性调控作用。方法应用PCR技术，从基因组DNA中扩增出hMAM启动子／增强子DNA序列，构建于PGL3报告基因上游，分别转染体外培养的乳腺癌细胞MDA—MB-415、T47D及胃癌细胞7901，分析启动子和增强子序列对乳腺癌细胞的基因表达调控作用。结果酶切图谱分析、DNA序列测定表明成功构建hMAM启动子／增强子调控的表达载体；荧光素酶报告基因检测结果分析表明，hMAM启动子／增强子能够调控报告基因的表达。结论hMAM启动子／增强子，在MDA—MB-415乳腺癌细胞具有调控基因表达的作用；

Construction of expression vector controlled by hMAM promoter and enhancer

Objective To construct expression vector under control by hMAM promoter and en- hancer of hMAM and to explore their function on specific regulation in breast cancer cells. Methods hMAM promoter and enhancer DNA fragments were amplified by PCR from genomic DNA and reconstructed on the upstream of reporter gene in the PGL3 vector. Breast cancer cells （ MDA-MB- 415, T47D） and gastric cancer cells （7901） in vitro were transfected with the constructs for analyzing the efficacy and specificity of the hMAM promoter and enhancer. Results Restriction enzymes analysis and DNA sequencing showed that the recombinant plasmids were corrected. Luciferase reporter assay proved that hMAM promoter and enhancer fragments could regulate the expression of report gene. Conclusion hMAM promoter and enhancer fragments possess regulatory effect on gene expression in breast cancer MDA-MB-415 cells.