黄热病毒特异性抗原片段的筛选与鉴定

目的通过系统的生物信息学分析和实验验证,筛选黄热病毒的特异抗原片段,为免疫诊断试剂的研制奠定基础。方法分别利用DNAStar及ANTHEPROT软件对黄热病毒蛋白进行分析,参考亲水性、抗原性、可塑性、表面可及性及二级结构信息,对黄热病毒可能的共有及特异性抗原表位进行系统的预测分析,分析其在不同毒株中的保守性,并对预测得分值较高的抗原区域进行RT-PCR扩增,利用pMal-c2x原核表达系统进行原核表达,Western blot验证其免疫学反应原性,检测阳性抗原片段经亲和纯化后,包被ELISA微孔板,进一步验证其免疫学反应特异性及检测敏感性。结果其中27段抗原片段获高效表达,经Western blot筛选及ELISA进一步验证,原核表达抗原片段YFV22表现良好特异抗,可检测低至1∶12 800倍稀释的黄热病毒多克隆抗体,与所试其他参考病毒多克隆抗体无交叉反应。结论经系统筛选、验证,获黄热病毒特异性抗原片段1段,为其免疫学诊断试剂的研制奠定了基础。

Screening and identification of yellow fever virus-specific antigens

Objective To screen and identify yellow fever virus specific antigens for the diagnosis of the disease.Methods The bioinformatic software DNAstar and ANTHEPROT were used to analyze the features of proteins including their hydrophilicity,flexibility,surface probability and antigenicity and their secondary structures,those possible shared and specific antigen epitopes were predicted and analyzed systematically especially for their conservation in different strains.Based on the result of bioinformatic analysis,some antigen epitopes were amplified and inserted into prokaryotic expression vector pMal-c2x which was then transferred into Rosetta（DE3）.Isopropyl-β-D-thiogalactoside（IPTG）and SDS-PAGE were used to induce the expression and-dentification of proteins.Then the antigenicity of expressed proteins was determined by Western blot.Results 27 epitopes were efficiently expressed in E.coli and YFV22 segment was found to be highly specific and sensitive which could detect very low level of monoclonal antibody and also had no cross reaction with other viruses.Conclusions Antigen fragment YFV22 specific to yellow fever virus is obtained and can be used to develop immunological diagnostic reagents.