E-钙黏蛋白和N-钙黏蛋白启动子的构建及活性测定

Construction and activity characterization of E-cadherin and N-cadherin promoters

目的构建含有E-钙黏蛋白(cadherin)基因和N-钙黏蛋白基因启动子的荧光素酶报告基因载体,并检测E-钙黏蛋白启动子和N-钙黏蛋白启动子的活性。方法利用PCR技术从乳腺癌细胞ZR75-1基因组中扩增出E-钙黏蛋白和N-钙黏蛋白启动子片段,插入到荧光素酶报告基因载体p GL4-basic中,确定所扩增的DNA序列后,将其转染至乳腺癌细胞ZR75-1和肝癌细胞Hep G2中,检测启动子的活性。结果测序结果表明,扩增的E-钙黏蛋白启动子序列正确;荧光素酶活性实验表明,E-钙黏蛋白启动子和N-钙黏蛋白启动子在乳腺癌细胞ZR75-1和肝癌细胞Hep G2中表达都具有活性。结论成功构建了E-钙黏蛋白启动子和N-钙黏蛋白启动子报告基因表达载体,为进一步筛选调节E-钙黏蛋白和N-钙黏蛋白表达的转录因子提供了重要基础。

Objective To clone E-cadherin and N-cadherin promoters and insert them into a luciferase reporter gene vector, and to characterize the promoter activity of E-cadherin and N-cadherin. Methods E-cadherin and N-cadherin pro- moter were cloned into pGIA-basie. The resulting plasmids were determined by DNA sequencing. The promoter activity was analyzed in breast cancer cell line ZR75-1 and hepatocareinoma cell line HepG2. Results DNA sequencing showed that the sequences of the cloned promoter regions were correct. Analysis of the reporter gene activity indicated that the E-cad- herin and N-cadherin promoters had the highest transcriptional activity in ZR75-1 and HepG2 cells. Conclusion The E- cadherin and N-cadherin promoter genes are cloned successfully, contributing much to screening transcription factors that regulate E-cadherin and N-cadherin expression.