新型雌激素受体β信号通路调节因子的分离及鉴定

Isolation and analysis of a novel co-regulator of estrogen receptor beta

目的:利用酵母双杂交技术筛选与ERβ相互作用的蛋白,为进一步研究ERβ的功能奠定基础。方法:从乳腺cDNA文库中钓取ER-βAF1 cDNA,构建诱饵蛋白载体pAS2-1-ERβ-AF1,利用酵母双杂交筛选技术,在乳腺cDNA文库中筛选与ERβ-AF1相互作用的蛋白。将含有pACT2-候选基因的酵母菌与含有pAS2-1-ERβ-AF1的酵母菌进行交配实验,利用LacZ报告基因检测ERβ-AF1与候选蛋白之间是否存在相互作用。然后将ERβ-AF1和候选蛋白分别构建到pGEX-KG和pET-28 a上,并纯化GST-ERβ-AF1和H is-候选蛋白融合蛋白,利用GST沉淀实验进一步验证ERβ-AF1和候选蛋白的体外相互作用。结果:所获取的候选蛋白为CTGF,是CCN多肽家族成员。交配实验表明,CTGF与ERβ-AF1特异相互作用,而不与空载体或BRCT1对照相互作用。GST沉淀实验进一步验证,CTGF与ERβ、ERα均存在相互作用,但与CTGF同源性较高的NOV蛋白却不与ERβ、ERα结合。结论:CTGF和ERβ存在特异的相互作用。

Objective：To isolate and characterize proteins interacting with estrogen receptor β. Methods： The cDNA encoding ERβ-AF1 was isolated from human breast cDNA library and cloned into the bait protein expression plasmid pAS2-1. The standard yeast two-hybrid screen was pedormed to isolate proteins interacting with ERβ-AF1. To further validate the interaction, using the LacZ as a reporter gene, the mating test was performed between the yeasts containing pACT2-candidate gene and pAS2-1-ERβ-AF1. ERβ-AF1 and candidate gene were then cloned into pGEX-KG and pET-28a, respectively. The fusion proteins GST-ERβ-AF1 and His-tagged candidate protein were purified through affinity column. The GST pull-down assay with the purified proteins further confirmed the interaction. Results： The candidate protein was identified as CTGF,a member of CCN polypeptide super-family. The mating test indicated that ERβ-AF1 interacted with CTGF but not with empty vector or BRCT1. GST pull-down assay further demonstrated that CTGF, but not NOV, a highly homologous member of CCN polypeptide super-family that shares high homology with CTGF, interacted with both ERβ and ERα. Conclusion： ER interacts with CTGF specifically in yeast cells and in vitro.