重组人组蛋白H3和H4的表达纯化及其细胞毒性分析

发布时间：2018-03-19 03:02

本文选题：重组人组蛋白　切入点：人血清白蛋白　出处：《中国生物化学与分子生物学报》2016年02期 　论文类型：期刊论文

【摘要】：细胞外组蛋白在脓毒症、类风湿性关节炎、急性肺损伤等多种疾病的发生发展中起关键作用,但由于缺乏合适的标准品,至今无法对患者体内的胞外组蛋白进行精确定量,导致在多种感染性疾病中无法根据血清组蛋白含量对疾病进行精确分级,也无法据此合理用药。同时,对患者体内胞外组蛋白精确定量也有助于确定细胞毒性机制研究的使用剂量。本研究用大肠杆菌表达单体变性组蛋白H3和H4,亲和纯化后用梯度稀释和透析方法,可以得到复性的组蛋白单体H3、H4以及H3/H4复合物。通过对蛋白质在纯化过程中稳定性的比较,发现H3/H4复合物较单体更为稳定。以该复合物(50μg/m L)处理HUVEC细胞,细胞存活率约为20%,与小牛胸腺组蛋白(200μg/m L)的毒性类似。该复合物引起的细胞毒性可被人血清白蛋白以浓度依赖的形式(0.625~10 mg/m L)缓解,提示其构象基本正确。因此,重组组蛋白H3/H4复合物可以作为精确定量组蛋白的标准品,对基于组蛋白含量的疾病分级和组蛋白毒性机制的研究均有应用价值。
[Abstract]:Extracellular histone plays a key role in the development of many diseases, such as sepsis, rheumatoid arthritis, acute lung injury and so on. In many infectious diseases, it is impossible to classify the disease accurately according to the content of serum histone, and it is impossible to use the medicine reasonably according to the level of serum histone. At the same time, Accurate quantification of extracellular histone was also helpful to determine the dose of cytotoxic mechanism. We used Escherichia coli to express denatured histone H3 and H4 and purified them by gradient dilution and dialysis. By comparing the stability of protein during purification, we found that H3 / H4 complex is more stable than monomers. The H3 / H4 complex was used to treat HUVEC cells with 50 渭 g / mL of H3 / H4 complex. The cell survival rate is about 20%, which is similar to the toxicity of calf thymus histone 200 渭 g / mL. The cytotoxicity induced by this complex can be alleviated by human serum albumin (HSA) in a concentration-dependent manner, suggesting that its conformation is basically correct. The recombinant histone H3 / H4 complex can be used as a standard for accurate quantification of histone. It is valuable for the study of histone classification and the mechanism of histone toxicity based on histone content.
【作者单位】：上海交通大学医学院附属上海儿童医学中心卫生部儿童血液肿瘤重点实验室;上海交通大学医学院附属上海儿童医学中心儿科转化医学研究所感染科;
【基金】：国家自然科学青年基金(No.81201450) 上海市科委启明星计划(No.14QA1402900) 上海市博士后资助项目基金(No.2014M551418)~~
【分类号】：R329.2

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