中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (10): 705-707.

• 论著 • 上一篇    下一篇

SLE患者基因组DNA甲基化水平的检测

朱小华1,李锋2,陈国梁3,杨永生1,林尽染4,徐金华5,项蕾红6   

  1. 1. 复旦大学附属华山医院
    2. 上海市复旦大学医学院附属华山医院皮肤科
    3. 上海复旦大学附属华山医院皮肤科
    4. 复旦大学附属华山医院皮肤科
    5. 上海市复旦大学附属华山医院皮肤科
    6. 上海华山医院皮肤科
  • 收稿日期:2008-09-22 修回日期:2008-10-31 发布日期:2009-10-09
  • 通讯作者: 朱小华 E-mail:alphabetist@163.com
  • 基金资助:

    市(厅)级基金（编号）

DNA methylation in peripheral blood mononulclear cells from patients with systemic lupus erythematosus

  • Received:2008-09-22 Revised:2008-10-31 Published:2009-10-09

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摘要：

目的 构建甲基CpG结合域（MBD）蛋白检测分析SLE患者基因组DNA甲基化水平。方法将重组表达质粒MBD-pET30b+转化大肠杆菌DH5a克隆扩增并测序鉴定后，转化大肠杆菌BL21（DE3）后接种于LB 培养基中,异丙基硫代半乳糖苷（ IPTG）诱导重组蛋白的表达, 表达产物包涵体经Ni－NTA亲和层析纯化，通过SDS－PAGE和Western印迹鉴定蛋白表达。用此蛋白免疫染色人胚肾细胞后用荧光显微镜观察、免疫染色正常人（15例）和SLE患者（21例）外周血单核细胞后用流式细胞仪检测平均荧光强度。结果 克隆质粒测序结果与理论预期完全一致，SDS－PAGE显示在大肠杆菌中表达出分子量为46 000的目标蛋白，Western印迹显示目标蛋白带有His融合标签（氨基端）和HA标签（羧基端）。荧光显微镜观察显示MBD蛋白能与细胞内的甲基化CpG基序特异性结合，流式细胞仪检测显示SLE患者DNA甲基化水平（9.32 ± 1.33）明显低于正常人（11.66 ± 1.04）（P < 0.05），SLE患者DNA甲基化水平与SLE-DAI负相关（r = －0.78，P < 0.01）。结论 SLE患者DNA甲基化水平明显低于正常人，并与SLE-DAI负相关。

关键词: 甲基CpG结合域, 原核表达, DNA甲基化, 红斑狼疮，系统性

Abstract:

Objective To assess the DNA methylation status in peripheral blood mononulclear cells （PBMCs） from patients with systemic lupus erythematosus （SLE） using a constructed methyl-CpG-binding domain （MBD） proteins. Methods A recombinant plasmid MBD-pET30b+ was transformed into E. coli （DH5a） for clonal expansion followed by sequencing. Then, plasmids with correct sequence were transformed into E. coli BL21 （DE3） for the expression of MBD proteins under the induction of isopropy-β-D-thiogalactoside （IPTG）. The expression products were purified by Ni-NTA chromatography and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） and Western blotting. Cultured 293T cells and isolated PMBCs from 21 patients with SLE and 15 normal human controls were immunostained with the expressed protein, and analyzed by fluorescence microscopy and flow cytometry. Results The sequence of recombinant plasmid was proved to be consistent with the expectation. SDS-PAGE and Western blotting demonstrated that the molecular weight of expressed tetrameric-protein was 46000, with the presence of N-terminal His-tag and C-terminal HA-tag. As fluorescence microscopy showed, the MBD proteins could specifically bind to intracellular CpG DNA in 293T cells. Patients with SLE had a significantly lower methylation level than the controls did （9.32 ± 1.33 vs 11.66 ± 1.04, t = 5.68, P < 0.05）, and the DNA methylation level negatively correlated with SLE disease activity index （r = -0.78, P < 0.01）. Conclusions There is a lower DNA methylation in patients with SLE than in normal human controls, and the methylation level negatively correlates with SLE disease activity index.