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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

23卷

期数:

2017年01期

页码:

28-31

栏目:

基础医学

出版日期:

2017-01-16

文章信息/Info

Title:

Research on the activity of MDR1 promoter

文章编号:

1006-8147（2017）01-0028-04

作者:

刘 蕊

（天津医科大学第二医院药剂科，天津300211）

Author(s):

LIU Rui

（Department of Pharmacy, The Second Hospital, Tianjin Medical University, Tianjin 300211,China）

关键词:

多药耐药基因-1; 启动子; 荧光素酶报告基因; 活性鉴定

Keywords:

MDR1;  promoter;  luciferase reporter gene;  detection of activity

分类号:

R965

DOI:

-

文献标志码:

A

摘要:

目的：克隆编码多药耐药基因-1（MDR1）中不同长度的启动子片段并探讨和比较其在乳腺癌细胞中的转录活性。方法： 利用PCR扩增技术以MCF-7/ADR细胞基因组为模板克隆3种不同长度的启动子片段。pGL3-basic的启动区域中，构建一系列报告基因载体pGL3-MDR1Pn。以pGL3-control为阳性对照，分别将含不同长度的MDR1启动子的报告基因载体pGL3-MDR1Pn与pRL-TK以一定比例共转染到敏感的MCF-7细胞和耐阿霉素的MCF-7/ADR细胞，通过分析表达的荧光素酶的活性从而比较不同长度的启动子片段在这两种细胞中的转录活性。结果：通过酶切鉴定和测序验证了已将不同长度的MDR1启动子片段成功插入到荧光素酶报告基因载体pGL3-basic中，且克隆的片段中没有出现碱基突变。将表达载体转染细胞后活性检测结果显示pGL3-MDR1P1、pGL3-MDR1P2和pGL3-MDR1P3在MCF-7中活性分别为阳性对照的(13.03 2.35)%、(14.60 3.57)%和(10.27 1.89)%；而在MCF-7/ADR中活性分别为阳性对照的(105.26 6.84)%、(59.08 4.95)%和(62.39 5.76)%。结论：成功克隆了MDR1启动子片段，并成功构建了荧光素酶报告基因载体pGL3-MDR1P。启动子的活性分析结果初步表明长度约为2 000 bp的MDR1P1在MCF-7/ADR中具有相对较高的特异性。

Abstract:

Objective: To clone different lengths of promoter from multi-drug resistance gene 1 and analysis its activities in MCF-7 cells or MCF-7/ADR cells. Methods: According to a review about the transcriptional regulation to human multi-drug resistance gene 1, three fragments of MDR1 promoter from MCF-7/ADR cell were amplified by PCR, and then they

作者简介 刘蕊（1989-），女，药师，学士，研究方向：临床药学； E-mail：Li351227007@163.com。

were inserted into pGL3-basic, a luciferase reporter gene vector, to construct pGL3-MDR1Pn which were directed by MDR1 promoter. The different lengths of MDR1 promoter reporter gene vector pGL3-MDR1Pn and pRL-TK were co-transfected into MCF-7 cells or MCF-7/ADR cells in a certain proportion, where pGL3-control was a positive control. The transcriptional activity of MDR1 promoter at three different lengths in the two kinds of cells were compared by the luciferase activity. Results: The constructions of pGL3-MDR1Pn were determined by DNA sequencing. Compared with the positive control, the activities of pGL3-MDR1P1, pGL3-MDR1P2 and pGL3-MDR1P3 in MCF-7 were (13.03 2.35)%, (14.60 3.57)% and (10.27 1.89)%, respectively. But their activities in MCF-7/ADR were (105.26 6.84)%, (59.08 4.95)% and (62.39 5.76)%, respectively. Conclusion: The MDR1 promoter is cloned successfully and the luciferase reporter gene vectors pGL3-MDR1P are successfully constructed. These results show that MDR1P1 with 2000 bp may have higher specificity in MCF-7/ADR cells than that in MCF-7 cells.

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