摘要
目的：本研究旨在利用细菌表面展示技术结合流式细胞术建立一种快速直观的抗原表位的新方法。方法：本研究以传染性法氏囊病毒结构蛋白VP2作为目标蛋白，对该方法的可行性进行检测。将VP2基因分为连续不重叠的5个基因片段（V1～V5）。将各段基因分别克隆至细菌展示载体pAPEx中．转化大肠杆菌DH5a，利用IPTG诱导使蛋白片段锚定表达于大肠杆菌细胞间质侧的内膜上，利用溶菌酶破除细菌外膜（原生质球制备），使蛋白片段暴露于原生质球表面。将该原生质球先后与传染性法氏囊病毒多克隆抗体和FITC标记的兔抗鸡抗体进行孵育，利用流式细胞仪（Flowcytometer，FC）检测原生质球的荧光信号强度。结果：FC结果显示，除V2片段外其余片段均有很强的荧光信号，其荧光强度为V3最强，Vl、V4、V5其次。该结果说明V1、V3、V4、V5段存在抗原表位，V2段中无表位。为验证Fc结果，本研究利用传统方法对各基因片段进行表达、纯化及Westernblot鉴定，结果显示V3与多抗的结合能力最强，v2段无阳性反应，与FC结果相符。此外，本研究利用抗原表位分析软件对各蛋白片段潜在抗原表位进行预测，其结果与FC结果相一致。结论：本研究利用Fc对抗原表位筛选过程进行实时监测，通过荧光信号强弱即可判断抗原表位是否存在及强弱，与传统抗原表位筛选方法相比，节省了时间提高了效率。利用该方法可以快速地筛选出优势表位，对于流行病病原的基础研究及疫苗的研制具有重意义。
Objective：To establish a novel method with fast, high-throughput and visualized characteristics to identify epitopes of given antigen on the basis of bacterial display technology and flow cytometry （FC）. Methods： VP2 protein of infectious bursal dis- ease virus （IBDV） was used to testify the feasibility of this method as a target protein. VP2 gene was truncated into sequential and non- overlapping five gene fragments （ V1 -V5 ）. These fragments were cloned into a bacterial display carrier pAPEx and transfbrmed in to E. coil DH5oL. After inducing with IPTG, fragments of VP2 protein anchored and expressed on the bacterial inner membrane. After dis- ruption of the outer membrane by EDTA-lysozyme, spheroplasts anchored with VP2 fragments were incubated with purified chicken an- ti-IBDV polyclonal antibody （pAb） and FITC-rabbit anti-chicken IgG. FC was utilized to detect the fluorescence intensity of sphero- plasts. Results ： The results of FC showed that fragments except V2 have strong fluorescence intensity and the fluorescence intensity of V3 was the strongest of all. It indicated that dominant epitopes primarily localized in V1, V3, V4 and VS, and barely in V2. To dem- onstrate the results of FC, this study adopted traditional strategy that five truncated fragments were expressed, purified and identified by Western blot. The results of Western blot indicated that affinity of V3 and pAb was the strongest and V2 had no reaction with pAb which was consistent with FC. Otherwise, this research forecasted potential epitopes of fragments using analysis software and it＇ s ac- cordant with FC. Based on experimental data above, we could draw a conclusion that it＇ s feasible to screen epitopes using FC. The u- tilization of FC drove the process of screening epitopes in a real-time way, and through fluorescence intensity we could judge whether epitopes exist in the target fragments or not. Conclusion ： This method dispenses with the process of expression and purification of pro- teins and detection of immunogenicity. It not only saves time but also enhances the efficiency. The application of this method can screen epitopes in a short time and it＇ s significant to the fundamental research of epidemic pathogen and development of vaccine.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2014年第3期366-372,共7页
Chinese Journal of Immunology
