比格犬雌激素受体ERβ以及剪切异构体ERβ_(419)、ERβ_(431)的转录活性分析

Transcriptional Activity Analysis of Beagle ERβ and Splice Isoforms ERβ_(419),ERβ_(431)

目的分析比格犬雌激素受体ERβ以及剪切异构体ERβ_(419)、ERβ_(431)的转录活性。方法将细胞分为6组,空白对照组(正常含有培养液的293T细胞),LV5-ERβ组(稳定表达ERβ的293T细胞),LV5-NC组(感染慢病毒LV5空载体的293T细胞),Lenti-OE-Flag-ERβ_(431)组(稳定表达ERβ_(431)的293T细胞),Lenti-OE-Flag-ERβ_(419)组(稳定表达ERβ_(419)的293T细胞),Lenti-OE-Flag-ERβ_(419)-NC组(感染慢病毒Lenti-OE-Flag空载体的293T细胞)。其中每组组内又分为2组,每组细胞瞬时转染构建的重组质粒ERE-LUC和RLUC,每组中的其中一组加入终浓度为10μmol/L的E_2,而另一组内加入等量无水乙醇。分别检测每组加入刺激(E_2和无水乙醇)前后的荧光素酶活性。结果成功地将雌激素反应元件ERE克隆到荧光素酶报告载体pGL3-Vector启动子上;设计引物扩增出的hRluc-neo基因成功克隆到pGL3-Basic载体;加入雌激素后,比格犬ERβ转录活性明显升高(P<0.01),而ERβ_(419)和ERβ_(431)转录活性在雌激素加入前后变化不大(P>0.05)。结论成功建立了比格犬ERβ的转录激活系统,而ERβ_(419)和ERβ_(431)的LBD区域都有部分的缺失,使得其不能与雌激素结合,继而转录活性无法被激活。

Objective To detect the transcriptional activity of ERβ,ERβ419 and ER431 expressed vector.Method The cells were divided into 6 groups,control groups,LV5-ERβ groups,LV5-NC groups,Lenti-OE-Flag-ERβ431 groups,Lenti-OE-Flag-ERβ419 groups,Lenti-OE-Flag-NC groups.Each group was further divided into two groups,each group of cells was transiently transfected with the recombinant plasmid ERE-LUC and RLUC.In each one group with a final concentration of 10 μmol/L E2,while the other group with an equal volume of anhydrous ethanol.The luciferase activity of each group was tested after adding the stimulation.Result ERE-LUC and RLUC recombinant plasmid was constructed succesfully.Compared with anhydrous ethanol group,The transcriptional activity of Beagle ERβ with estrogen treatment was significantly increased（ P 〈 0.01）,while there was no significant change neither ERβ419 nor ERβ431（ P 〉 0.05）.Conclusion Succesfully constructed transcriptional activation system of Beagle ERβ,but the partly lack of LBD lead to ERβ419 and ERβ431 can ’t combine with E2,then transcriptional activity could not be activated.