人乳腺癌相关抗原DF3转录调控序列的克隆及其调控表达

Cloning and regulatory expression of the transcriptional regulatory sequences in human breast cancer related DF3 antigen

目的 克隆人乳腺癌相关抗原DF3转录调控序列 (TRS) ,并检测其活性与细胞表面抗原DF3之间的关系。方法 通过PCR技术克隆出人乳腺癌相关DF 3抗原 5′端 771bp的转录调控序列。分离纯化PCR产物并与 pMD 18 T载体相连接 ,酶切 ,测序验证。将DF 3TRS克隆到pGL3报告载体中。瞬时转染DF3阳性乳腺癌细胞株MCF 7,DF 3阴性乳腺癌细胞株MDA MB 2 3 1。荧光素酶定量分析检测启动子的活性。结果 酶切 ,测序结果验证所获得的序列正确 ,在MDA MB 2 3 1中只能检测到背景水平的荧光 ,而在MCF 7中荧光水平约为MDA MB 2 3 1的 2 0 0倍。结论 成功克隆出人乳腺癌相关抗原DF3转录调控序列 ,其活性与乳腺癌相关抗原DF 3存在着明显的相关性 。

Objective To clone the transcriptional regulatory sequences (TRS) in human breast cancer related DF3 antigen, and to test the relationship between the activity of the TRS and the cell surface DF3 antigen. Methods Authors designed a pair of primers according to the registered 5′-flanking region of DF3 antigen. The 771 base pairs of DNA fragment were amplified from the genomic DNA of human MCF-7 breast carcinoma cells by PCR, and cloned to the pMD18-T vector. The results were tested by restrictive enzyme analysis and DNA sequencing. The DF3 TRS was cut by double enzyme: Mlu I、Hind III,and cloned to the pGL3 vector . The activity of the DF3 TRS was expressed by analyzing the relative luciferase activities. Results Restrictive endonuclease identification and DNA sequencing proved that the sequence authors got, was correct. The luciferase activity in MDA-MB-231 was hardly detected, whereas in MCF-7 the luciferase activity was about 200 times than in MDA-MB-231. Conclusions The DF3 TRS was cloned successfully. The DF3 activity has a distinct relationship with DF3 antigen. The study shows that DF3 TRS can be used in the gene therapy of breast carcinoma.