斑点杂交法检测人群外周血白细胞中泛素化组蛋白H2B

Detection of ubiquityl-histone H2B in human peripheral blood leukocytes by dot blot hybridization

目的建立一种简单、快速、准确地检测人群外周血白细胞中泛素化组蛋白H2B（ubiquityl．histoneH2B，ubH2B）修饰水平的方法。方法利用ubH2B标准样品建立检测人群外周血白细胞中ubH2B的斑点杂交方法，确定最佳的封闭液及一抗、二抗稀释度，并确定斑点杂交方法的灵敏度、检测范围及重复性。采用建立的斑点杂交方法检测45份人外周血白细胞中ubH2B的水平。结果斑点杂交方法检测ubH2B的最佳封闭液为5％牛血清白蛋白（albuminfrombovineserum，BSA），一抗稀释度为1：500、二抗稀释度为1：10000。斑点杂交方法检测ubH2B的灵敏度为150ng。ubH2B浓度在3．000～48．000ng／p,1有良好线性关系，决定系数（R2）=0．998。批内和批间的变异系数分别为7．00％和6．08％。45份人群组蛋白样品中ubH2B为0～4．090ng／wl，ubH2B在总组蛋白中的含量范围为0～255．625ng／mg。结论斑点杂交方法灵敏度高、重复性好，可应用于人群外周血白细胞中ubH2B的检测。

Objective To establish a simple, rapid and accurate method for detection of ubiquityl-histone H2B （ubH2B） in human peripheral blood leukocytes. Methods UbH2B polypeptide was utilized to establish the dot blot hybridization method for detection of ubH2B in peripheral blood leukocytes. The optimal blocking buffer and the diluted concentration of primary and secondary antibodies were determined by dot blot hybridization. And the sensitivity, detection range and repeatability of dot blot hybridization were determined. Meanwhile, the levels of ubH2B in 45 people peripheral blood leukocytes were quantified by the established dot blotting hybridization. Results The optimal conditions of dot blot hybridization for detection of ubH2B were the blocking buffer of 5% albumin from bovine serum （BSA）, a diluted concentration of primary antibody of 1 ： 500 and a diluted concentration of secondary antibody of 1 ： 10 000. The sensitivity of dot blot hybridization was 150 ng. At the concentration range of 3.000 - 48.000 ng/μl, there was a good linear relation, and determination coefficient （R2） = 0.998. The coefficient of variation of the intra-assay and inter-assay was 7.00% and 6.08%, respectively. The detect results of 45 people samples were the concentration of ubH2B in histone samples ranging from 0 - 4.090 ng/μl, the amounts of ubH2B in total histone ranging from 0 - 255.625 ng/mg. Conclusion Due to a higher sensibility and better repeatability, dot blot hybridization can be applied to detect the ubH2B in human peripheral blood leukocytes.