幽门螺杆菌鞭毛蛋白FliD免疫学特征的初步研究

A preliminary study on the immunological characteristics of Helicobacter pylori flagellin, FliD

目的初步研究幽门螺杆菌(Helicobacter pylori,HP)鞭毛蛋白FliD的部分免疫学特征。方法原核表达重组FliD蛋白;制备rFliD兔多克隆抗体,同时酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测rFliD特异性免疫球蛋白G(immunoglobulin G,IgG)与免疫球蛋白M(immunoglobulin M,IgM)水平;酶联免疫斑点试验(enzyme-linked immunospot,ELISPOT)检测rFliD刺激Hp患者外周血单核细胞(peripheral blood monocytes,PBMC)后的γ干扰素(interferon-γ,IFN-γ)及白细胞介素4(interleukin4,IL-4)表达水平;流式细胞术(flow cytometry,FCM)检测rFliD蛋白刺激胃粘膜上皮细胞(Gastric epithelial cells,GES-1)细胞后的凋亡比例。结果rFliD原核表达纯化成功;成功制备高效价rFliD兔多克隆抗体,并发现rFliD可诱导家兔产生高水平IgG与IgM,与免疫前血清比差异具有统计学意义(t值分别为5.42、8.18,P值均<0.05)),效价可达1:102400;与健康对照组(healthy control,HC)相比,HP组rFliD特异性IFN-γ与IL-4均显著增高(t值分别为21.91,13.57,P值均<0.05),但产生IFN-γ的水平显著高于IL-4(t=7.55,P<0.05);与磷酸盐缓冲液(phosphate buffered saline,PBS)对照组相比,rFliD刺激GES-1细胞后24或72 h均可发生显著性凋亡且差异具有统计学意义(t值分别为10.12、10.90,P值均<0.05)。结论幽门螺杆菌鞭毛FliD蛋白具有强免疫原性,可诱导产生较强的体液及细胞免疫应答,且细胞免疫应答以IFN-γ高表达的Th2型为主;同时发现其对GES-1细胞存在一定程度的凋亡效应。

Objective To preliminarily study the partial immunological characteristics of Helicobacter pylori(HP)flagellin FliD.Methods FliD protein was acquired by prokaryotic expression.rFliD rabbit polyclonal antibody was prepared,the detection of rFliD-specific immunoglobulin G(IgG)and immunoglobulin M(IgM)antibody levels were performed by Enzyme-linked immunosorbent assay(ELISA)method.Enzyme-linked immunospot(ELISPOT)method was used to detect the expression levels of interferon-γ(IFN-γ)and interleukin-4(IL-4)released by peripheral blood monocytes(PBMC)which were stimulated by rFliD in HP patients.Flow cytometry(FCM)method was used to detect the proportion of apoptosis in GES-1 cells(Gastric epithelial-1 cells)stimulated by rFliD protein.Results The prokaryotic expression and purification of rFliD was successful.High titer rFliD rabbit polyclonal antibody was prepared successfully,and it was found that rFliD can induced high levels of IgG and IgM antibody in rabbit,compared with the pre-immune serum,there was a statistical difference(T values were 5.42 and 8.18,respectively,both P values<0.05,)and the antibody titer can reach at 1:102400.Compared with the healthy control(HC),rFliD specific IFN-γand IL-4 in HP was significantly higher than HC(T values were 21.91 and 13.57,respectively,both P values<0.05),and the level of IFN-γproduction was significantly higher than IL-4(t=7.55,P<0.05).The GES-1 cell apoptosis proportions of rFliD stimulated were significant higher than PBS(phosphate buffered saline),both at the time of 24 and 72 h(t values were 10.12 and 10.90,respectively,both P values<0.05).Conclusion Helicobacter pylori flagella FliD protein has strong immunogenicity and can induce strong humoral and cellular immune responses,and the cellular immune response is dominated by Th2 type with high expression of IFN-γ.At the same time,rFliD also can induced a certain degree of GES-1 cells apoptotic effect.