| 染色质体外组装相关蛋白的表达纯化及组装体系的建立 |
| 投稿时间：2011-06-21 修订日期：2011-11-20 点此下载全文 |
| 引用本文：赵忠亮,赵吉成,代辉,沈珝琲,张业.染色质体外组装相关蛋白的表达纯化及组装体系的建立[J].医学研究杂志,2012,41(1):19-23 |
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| 基金项目:国家自然科学基金资助项目（30871382）；教育部博士点基金资助项目（20101106110023） |
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| 中文摘要:目的提取并纯化真核细胞核内组蛋白，表达并纯化重组组蛋白、染色质组装相关因子NAP-1（nuclear assembly protein-1）及ACF（ATP-utilizing chromatin assembly and remodeling factor），并建立无细胞体外染色质组装体系。方法在大肠杆菌BL21中表达并应用离子交换层析纯化组蛋白，从真核细胞HeLa细胞核中提取并应用羟基磷灰石纯化组蛋白八聚体，利用杆状病毒表达系统在昆虫细胞Sf9中表达并纯化组装相关因子，应用纯化得到的蛋白在适宜条件下进行染色质体外组装，并用微球菌核酸酶酶切检测。结果获得了高纯度的原核表达的组蛋白，真核细胞组蛋白八聚体及组蛋白特异伴侣NAP-1和ACF；建立了由质粒DNA、纯化的真核细胞核内重组组蛋白、重组NAP-1、重组ACF及ATP为核心成分的体外染色质组装的无细胞系统，经微球菌核酸酶检测组装成功。结论利用体外分离纯化的多种重组蛋白，可成功构建基因的染色质模板，为在体外染色质模板上进行基因转录的调控机制研究奠定了基础。 |
| 中文关键词:组蛋白 ACF NAP-1 染色质组装 |
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| Purification of Chromatin Assembly Associated Proteins and Reconstitution Chromatin |
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| Abstract:ObjectiveTo extract and purify HeLa histones, express and purify recombinant histones, NAP-1 and ACF. To reconstitute chromatin in cell-free systems. MethodsHeLa core histone octamers were purified from the chromatin pellet of whole cell extracts from HeLa cells using hydroxyapatite chromatography. Individual histone proteins were purified from inclusion bodies of BL21 by ion exchange chromatography, and mixed to renature. NAP-1 and ACF were purified using baculovirus expression system in Drosophila. Then chromatin was assembled using isolated proteins and DNA in proper system，and was identified by micrococcal nuclease digestion. ResultsPurified HeLa histone octamers, recombinant histones, NAP-1 and ACF were shown. The system of chromatin assembly in cell-free systems using these isolated proteins was established. The chromatin was digested to DNA ladder by micrococcal nuclease. ConclusionWe assembled chromatin templates in vitro. It would be the basis of researches on regulation of gene transcription on chromatin template in vitro. |
| keywords:Histone ACF NAP-1 Chromatin assembly |
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