登革病毒共有抗原片段的筛选与鉴定

SELECTION AND PRIMARY DISTINGUISHING OF ONE FRAGMENT OF DENGUE VIRUS SHARED ANTIGEN SEQUENCE

目的经生物信息学分析及实验验证,筛选登革病毒共有的特异性抗原片段,为其免疫学诊断试剂的研制奠定基础。方法参考亲水性、抗原性、可塑性、表面可及性及二级结构信息,对登革病毒1-4型可能共有的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,并对部分抗原表位进行高效原核表达,采用Western blot验证其反应原性。结果利用pET32a原核表达系统对6种病毒(登革1-4型病毒、流行性乙型脑炎病毒、黄热病毒)部分可能的抗原片段进行了高效表达;经Western blot筛选,获得一段登革1-4型病毒共有抗原片段DV1-2,与实验中所用其它黄病毒属及甲病毒属多克隆抗体无明显的交叉反应。结论 DV1-2为登革1-4型病毒共有的特异性抗原片段,可用于其免疫学诊断试剂的研制。

Objective To analyse the protein epitopes of dengue virus type 1-4,and to distinguish the shared specific epitopes,so as to provide the basis for developing immunological diagnostic reagents.Methods Bioinformatic software DNAstar was used to analyze the hydrophilicity,flexibility,surface probability and antigenicity of dengue virus type 1-4 protein amino acid sequence and also the influence of secondary structure.Based on the bioinformatic analysis,6 specific epitopes were amplified and inserted into prokaryotic expression vector pET32a.Then the vectors were transferred into Rosetta（DE3）.Isopropyl-β-D-thiogalactoside（IPTG） was used to induce the expression of gene segments.SDS-PAGE were used to identify the expression of proteins.Then the antigenicity was tested using Western blot.Results Six epitopes from Dengue virus type 1-4,Japanese encephalitis virus,and yellow fever virus were expressed in E.coli,respectively.One dengue virus shared antigenic fragment（DV1-2） from dengue virus type 1 was confirmed using Western blot.And there was no obvious cross reaction of DV1-2 with Japanese encephalitis virus,yellow fever virus,Chikungunya virus（CHIKV） and sindbis virus（SINV） polyclonal antibody.Conclusion Fragment DV1-2 showed a good reactogenicity,and can be used to develop immunological diagnostic reagents.