重组人组蛋白H3和H4的表达纯化及其细胞毒性分析

Expression and Purification of Recombinant Human Histone H3 and H4 for Cytotoxicity Analysis

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。

Histones normally exist in the cell nuclei,but are also released outside to the lumen forming so-called extracellular histones. Extracellular histones pose strong cytotoxicities to cells,particularly in the disease pathogenesis and progression of sepsis,acute lung injuries and rheumatoid arthritis. The quantification of extracellular histone levels in the patients ＇ plasma demands high quality histone standard. Otherwise,circulating histone levels will not be usable for the disease stage classification and evaluation of drug dosage. In laboratories,quantification of extracellular histones is used for exploring the mechanism of histone toxicity. In the present study, we expressed denatured histone H3 and H4 monomers in E. coli. After purification with affinity column,the refolded H3 and H4 monomers,as well as H3/H4 complex,could be obtained by either gradient dilution or dialysis. We found that the H3/H4 complex was the most stable form of recombinant histones. Treatment of HUVECs with 50 μg/m L recombinant H3/H4 complex induced about 80% cell death,equivalent to the treatment with 200 μg/m Lcalf thymus histones. Such cytotoxicity could be relieved dose-dependently with human serum albumin from 0. 625 ~ 10 mg/m L,which was similar to the situation with natural H3/H4 complex. Therefore,our recombinant H3/H4 complex was usable as a quantification standard for the study of histone-induced cytotoxicities.