UreB蛋白B细胞抗原表位快速筛选与鉴定

以幽门螺旋杆菌(Helicobacterpylori,Hp)主要抗原蛋白尿素酶B(ureaseB,UreB)为靶蛋白,建立一种新的B细胞抗原表位筛选与鉴定方法.运用Fmoc固相肽合成法合成11条HpUreB蛋白的单表位抗原肽片段,在其氨基端标记FITC荧光素,应用荧光偏振方法(fluorescencepolarization,FP)快速鉴定这些肽片段的抗原性,并通过FP法在大规模样品中快速筛选相应抗体滴度高、分布人群广的优势抗原表位肽.结果表明,合成的11条UreB蛋白线性抗原肽中,10条具有较强的抗原性,其中No.2、No.5和No.11抗原肽相应的特异性抗体在感染Hp的人群中分布较广,抗体滴度较高,为UreB的优势抗原表位肽.对抗原表位进行多参数综合分析与设计,通过FP技术快速鉴定抗原肽,并筛选优势抗原表位肽,对于疾病的抗原表位谱研究具有重要的意义,同时在疾病的诊断、分型及治疗中具有重要的应用前景.

Rapid Screening and Identification of Dominant B Cell Epitopes of UreB Protein by Fluorescence Polarization Assay

In order to develop a new method for screening and identification of dominant B cell epitopes using UreB protein as a target antigen 11 amino acid fragments from UreB protein of Helicobacter pylori (Hp) were synthesized by Fmoc solid phase peptide synthesis strategy, and fluorescein FITC was labeled to the N-terminals of all peptides respectively. The antigenicity of synthetic peptides is determined by analyzing the recognition and combination between peptides and standard antibody samples by fluorescence polarization (FP) immunoassay. In order to screen the dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against 10 UreB synthetic peptides respectively in 159 UreB antibody-positive antiserum samples. There are 10 of 11 UreB synthetic peptides have distinct antigenicity by FP assay. The results showed that 3 out of the 10 antigenic peptides may be immunodominant, for the antibodies against them existed more widely among the samples and the antibody titers were higher than those of other peptides's. The methods for predicting and identifying epitopes are useful for epitope mapping, and the fluorescence polarized method for antibody immunoassay can be widely used in the diagnosis, typing and therapy of diseases in clinic in the future.