模拟表位型EB病毒感染诊断抗原的制备

Preparation of peptide mimotope-based diagnostic antigen of Epstein-Barr virus infection

目的利用大肠埃希菌表达系统获取模拟表位型诊断抗原用于EB病毒（epstein—Barr virus，EBV）感染的早期诊断，并初步评价其抗原性。方法将EB病毒模拟表位GP125、F1、A2、A3C2多肽序列用特定连接臂串联，密码子优化后全基因合成；将目的片段插入带有Trx标签的表达质粒中，在大肠埃希菌中进行表达，并利用亲和层析和离子交换层析纯化目的蛋白；利用Western blot技术对目的蛋白抗原性进行鉴定，并建立EBV—IgM抗体检测捕获法ELISA技术，初步评价此方法对阴阳血清样本的鉴别能力。结果所获取EBV模拟表位诊断抗原浓度为2．8mg／ml，纯度为99．01％；Western blot实验发现，以anti-Trx单克隆抗体或EBV—IgM阳性血清作为第一抗体，在NC膜约26×10^3处都可以发现特异条带（与预期一致）；对50份EBV急性感染阳性血清和50份阴性血清进行检测得出，阳性血清样本OD值1．352（95％CI：1．233～1．489）与阴性血清样本OD值0．135（95％CI：0．113～0．159）分布区组明显（P〈0．05），其中灵敏度为98．0％，特异性为96．0％，一致性检验kappa值为0．940，一致性优异。结论原核表达与亲和及离子交换层析纯化可以获取抗原性良好的EBV感染诊断抗原，偶联HRP后可以作为检测抗原应用于EBV-IgM捕获法ELISA血清学检测中。

Objective To prepare peptide minotope-based recombinant diagnostic antigen of Epstein-Barr virus （EBV） infection and evaluate its antigenicity preliminarily. Methods With Trx at the N-terminal and His tag at the C-terminal, the peptide minotope of EBV （GP125, F1, A2, A3C2） was expressed in Escherichia coli and purified by affinity and anion exchange chromatography （designated ＇ H58 ＇ ） ; based on antigenicity of H58 identified by Western blotting （WB） , we constructed and evaluated a novel early diagnostic ELISA for EBV infection. Results The soluble H58 protein with high concentration （2.8 mg/ml） and purity （99.01） was obtained; WB analysis found that there was an obvious band （28 ×10^3 ） on the NC membrane, using H58 anti-Trx monoclonal antibody or acute-phase sera of EBV infection as the first antibody. With the novel ELISA, 50 positive sera of EBV infection and 50 negative sera were detected, displaying that the grouping of OD value of positive serum （95% CI： 1. 233-1. 489 ） and negative serum （95% CI：0. 113-0. 159） was different （ P 〈 0. 05 ） with the sensitivity 98.0% , specificity 96.0% and kappa value 0. 940. Conclusions By E. coli expression and affinity and ion exchange chromatography purification, the peptide minotope-based recombinant diagnostic antigen of EBV infection was obtained with excellent antigenicity, which could be applied for serological detection of EBV infection.