目的 对主要黄病毒结构蛋白的抗原表位情况进行系统分析,鉴别共同及特异性抗原表位并对可能的特异性抗原表位进行原核表达.方法 分别利用DNAStar及ANTHEPROT软件对登革病毒1～4型、流行性乙型脑炎(乙脑)病毒及黄热病毒E蛋白的亲水性、抗原性、可塑性、表面可及性和Garnier-Robson的二级结构进行分析,预测可能的抗原表位.在此基础上,根据表位位置和氨基酸序列的相似性,分析6种病毒的共有及特异性抗原表位,并参考GenBank中的序列信息,对预测的抗原表位进行比对,进一步分析抗原表位在不同病毒株中的保守性.然后利用pET32及pMAL-c2x系统对可能的特异性抗原表位进行高效原核表达、Western blot验证其抗原性.结果 经系统的生物信息学分析,共预测获得登革病毒1～4型、乙脑病毒及黄热病毒共有抗原表位15个,病毒特异性抗原表位47个.利用pET32及pMAL-c2x系统对6种病毒部分可能的特异性抗原表位进行了高效表达.Western blot结果显示登革病毒1型及2型表达抗原片段表现良好的抗原反应原性,并与试验中所用其他黄病毒及甲病毒多克隆抗体无明显的交叉反应.而所表达的登革病毒3型和4型、乙脑病毒及黄热病毒片段无抗原反应原性.结论 6条原核表达抗原片段经Western blot验证,获得登革病毒1型和2型特异性抗原片段.%Objective To analysis the E protein epitopes of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus and to distinguish the shared or specific epitopes among them. Methods Bioinformatic software DNAStar was used to analyze the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E prtein amino acid sequences. The influence of secondary structure was also considered. Based on the bio-informatic analysis of E protein epitopes, 6 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-c2x. The vectors was then transferred into E.coli BL21 (DE3) and Rosetta (DE3). Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of gene segments and SDS-PAGE were used identify the expression proteins. The antigenieity was tested, using Western blot. Results 15 shared epitopes and 47 specific epitopes were forecasted by bioinformatic analysis, and 6 specific epitopes from dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein were expressed in E.coli successfully. Two specific antigenic determinant from dengue virus type 1 and dengue virus type 2 were confirmed using Western blot, while the others epitopes shown no antigenic reaction property. Conclusion Two specific antigenic determinant were confirmed, under Western blot.
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