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.