Landarani Zahra, Falsafi Tahereh, Mahboubi Mohaddese, Lameh-Rad Behzad
Department of Biology, Alzahra University, Tehran, Iran ; Department of Biochemistry, Payam-Nour University, Tehran, Iran.
Department of Biology, Alzahra University, Tehran, Iran.
Iran J Microbiol. 2014 Oct;6(5):324-9.
An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important virulence factor associated with peptic ulcer and gastric cancer risks. The purpose of this study was to isolate the 34 KDa OMP of H. pylori and evaluate its immunogenicity in experimental animals for rapid detection of more virulent H. pylori isolates.
Sarcosine insoluble fraction of membrane proteins (OMPs) were prepared from 15 clinical isolates of H. pylori and their profiles were analyzed by SDS-PAGE. Two out of 15 isolates which demonstrated higher expression for apparent 34 KDa proteins were selected. Under optimal conditions, 34 KDa protein was recovered from 5% SDS-Agarose gel, purified and injected into the New Zealand white rabbits with Fruend's adjuvant in multiple stages with two weeks intervals. Collected antiserum was purified through affinity chromatography with Sepharose column and its titer was determined by ELISA. Specific immune response was demonstrated by Dot blot and western blotting methods.
The titer of antibody was determined about 1/3000 and western blotting demonstrated a 34 KD-protein. Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology.
High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals. Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.
幽门螺杆菌的一种外膜蛋白（OMP）即OipA，是一种与消化性溃疡和胃癌风险相关的重要毒力因子。本研究的目的是分离幽门螺杆菌的34 kDa OMP，并在实验动物中评估其免疫原性，以快速检测毒力更强的幽门螺杆菌分离株。
从15株幽门螺杆菌临床分离株中制备膜蛋白（OMPs）的肌氨酸不溶部分，并通过SDS-PAGE分析其图谱。从15株分离株中选择了2株，这2株对表观34 kDa蛋白的表达较高。在最佳条件下，从5% SDS-琼脂糖凝胶中回收34 kDa蛋白，纯化后分多个阶段、间隔两周用弗氏佐剂注射到新西兰白兔体内。收集的抗血清通过琼脂糖柱亲和层析纯化，其效价通过ELISA测定。通过斑点印迹和蛋白质印迹法证明特异性免疫反应。
抗体效价测定约为1/3000，蛋白质印迹显示有一条34 KD的蛋白条带。通过斑点印迹法对各种菌株进行该蛋白的存在性筛选，结果表明其在从病情较重患者分离出菌株中的表达更为频繁。
获得的多克隆抗体效价较高，表明该蛋白在实验动物中具有高免疫原性。在从病情较重患者分离出的菌株中检测到34 kDa OMP，提示该多克隆抗体可能用于检测毒力更强的幽门螺杆菌菌株。
