原癌基因LMO2短剪切模式的结合蛋白鉴定及功能分析  

Identification of interaction partners and function analysis of new splicing product of human LMO2 gene

作　　者：袁伟[1] 杨爽[1] 孙伟[1] 杜君[1] 翟春利[1] 王兆琦[1] 朱天慧[1] 

机构地区：[1]南开大学医学院分子遗传学实验室,天津300071

出　　处：《中华血液学杂志》2008年第5期325-328,共4页Chinese Journal of Hematology

摘　　要：目的研究原癌基因LMO2短剪切模式（LMO2-C）的结合蛋白在白血病细胞K562中的表达及功能分析。方法利用麦芽糖结合蛋白（MBP）沉淀技术和哺乳动物双杂交技术研究LMO2-C的结合蛋白在K562细胞中的表达；半定量RT-PCR检测过表达LMO2-C的K562细胞中红系发育的标志性基因GPA的表达水平。结果MBP原核表达系统成功表达并被纯化出可溶性MBP-LMO2-C结合蛋白，利用MBP沉淀技术验证了LMO2-C能与K562细胞中内源性的GATA-1、LDB1结合；哺乳动物双杂交试验进一步证明LMO2-C能与LDB1结合，而且其过表达能够抑制LMO2-L与LDB1的结合，抑制率达（81．13±0．68）％；半定量RT-PCR结果显示在过表达LMO2-C的K562细胞中，GPA基因相对表达水平下调（51．00±1．58）％。结论LMO2-C能结合K562细胞中内源性GATA-1、LDB1蛋白，并能下调红系发育的标志性基因GPA的表达。Objective To identify the interaction partners of a new splicing product of LMO2 gene （LMO2-C）, and study its function in K562 cells. Methods Maltose binding protein （MBP） pulldown and mammalian two-hybrid assay （MTHA） were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein （GPA） in K562 cells. Results MBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pulldown analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being （81. 13 ± 0.68 ） %. RT-PCR results showed that the expression level of GPA was reduced [ （ 51.00 ± 1.58 ） % ] in K562 cells while LMO2-C overexpressed. Conclusion LMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.

关 键 词：基因 LMO2-C GATA1 LIM结构域结合蛋白 麦芽糖结合蛋白沉淀 双杂交系统技术 

分 类 号：R686[医药卫生—骨科学]