盐透析体外组装核小体及检测方法  

Assembly and Detection Methods of Nucleosome by Salt Dialysis in vitro

作　　者：赵宏宇[1,2] 柴荣[2] 王成爱[2] 蔡禄[2] 

机构地区：[1]内蒙古大学物理科学与技术学院,呼和浩特010021 [2]内蒙古科技大学数理与生物工程学院,内蒙古包头014010

出　　处：《河南师范大学学报（自然科学版）》2014年第1期133-136,共4页Journal of Henan Normal University(Natural Science Edition)

基　　金：国家自然科学基金(61072129);内蒙古自然科学基金(2013MS0514);内蒙古自治区博士研究生科研创新项目(B20131012610);内蒙古科技大学创新基金(2010NC056)

摘　　要：核小体是构成真核生物染色质的基本结构单位,体内研究核小体及染色质结构受到诸多因素限制,体外重构核小体结构是研究与核小体及染色质结构相关课题的一种重要的方法手段.实验将ES1,CS1以及601DNA序列克隆到载体中,通过PCR大量扩增回收得到目的DNA条带,表达纯化了4种组蛋白且装配成组蛋白八聚体,在盐透析的条件下组装形成核小体结构,利用EB染色以及Biotin标记的方法分析检测了形成核小体的效率.结果显示,在盐透析的条件下,可以有效的组装形成核小体结构,而且随着组蛋白八聚体与DNA的比例增加,核小体的形成效率显著提高.本实验为核小体定位、染色质重塑及组蛋白变体等表观遗传学以及结构生物学领域的研究奠定一定的基础.The nucleosomes constitute the basic structural unit of eukaryotic chromatin. In vivo eukaryotic nucleosome formation will be subject to interference from a variety of factors, and in vitro assembling nucleosome structure by DNA and histones octamer is one of basic method to study the nucleosome positioning and chromatin structure. In this work, the ES1, CS1 and 601 DNA sequence are cloned into plasmid, and then the DNA sequences are obtained through using PCR and gel ex- traction method. The H2A, H2B, H3 and H4 histones have been assembled to form octamer. After a salt dilution method em- ploying to assembly nucleosome, nucleosome reconstituted using different DNA sequences was analyzed by EB staining and bio- tin labeling. The results show that the nucleosome structure can be effectively reconstructed in vitro and the formation efficiency of nucleosome was positively related to the ratio of octamer to DNA. The work laid the foundation for further study of epigenetic and structural biology, such as nucleosome positioning, chromatin remodeling and histone variants.

关 键 词：核小体定位 组蛋白 Biotin标记 

分 类 号：Q71[生物学—分子生物学]