FHL2与IGFBP-7存在直接相互作用

目的：利用酵母双杂交技术筛选FHL2的相互作用蛋白，为进一步研究FHL2的功能奠定基础。方法：从卵巢文库中钓取FHL2 cDNA，构建诱饵蛋白栽体pAS2-1-FHL2，利用酵母双杂交筛选技术，在卵巢文库中筛选与FHL2相互作用的蛋白。将含有pACT2-候选基因的酵母菌与含有pAS2-1-FHL2的酵母菌进行交配实验，利用LacZ报告基因检测FHL2与候选蛋白之间是否存在相互作用。将FHL2和候选蛋白分别构建到pGEX-KG和pET-28a上，并纯化GST-FHL2和His-候选蛋白融合蛋白，利用GST沉淀实验进一步验证FHL2和候选蛋白的体外相互作用。结果：所获取的候选蛋白为IGFBP-7，是胰岛素样生长因子结合蛋白（IGFBPs）家族中的一员。交配实验表明，IGFBP-7与FHL2能特异性地相互作用，而不与空载体或BRCT1对照相互作用。GST沉淀实验进一步验证，IGFBP-7能与FHL2结合，而不与GST结合。结论：FHL2和IGFBP-7在体内及体外均可发生特异性的相互作用。

Direct interaction between FHL2 and IGFBP-7

Objective: To isolate and characterize protein s interacting with four and half LIM domain-2(FHL2) and lay a foundation for fur ther study on the function of the FHL2. Methods: The cDNA enco ding FHL2 was isolated from human ovary library and cloned into the bait protein plasmid pAS2-1. The standard yeast two-hybrid screen was performed to isolate the protein interacting with FHL2. To further validate the interaction, using t he LacZ as a reporter gene, the mating test was performed between the yeast cont aining pACT2-candidate gene and pAS2-1-FHL2. FHL2 and candidate gene were clo ned into pGEX-KG and pET-28a, respectively. The fusion proteins GST-FHL2 and His-tagged candidate protein were purified through affinity column.The GST pull -down assay with the purified proteins further validated the interaction. Results: The candidate protein was identified as IGFBP-7,a member of insulin-like growth factor-binding protein (IGFBPs) family. The mating tes t indicated that FHL2 interacted with IGFBP-7 but not with empty vector or BRCT 1. In vitro GST pull-down assay further demonstrated that FHL2 interacted w ith IGFBP-7 but not GST. Conclusion:FHL2 interacted with IGFB P-7 specifically in vivo and in vitro.