乙型脑炎病毒E蛋白抗原优势区域的鉴定

流行性乙型脑炎（Japanese Encephalitis，JE）是一种人与动物共患的虫媒病毒性疾病，严重危害着人畜的健康。E蛋白是乙型脑炎病毒（Japanese Encephalitis Virus，JEV）的主要结构蛋白，在病毒的吸附、融合、血凝、细胞趋向性、病毒毒力和诱导保护性免疫反应中起重要作用。为了确定该蛋白的抗原优势区域，本研究设计了3对引物，将E蛋白分成3个大段，分别是EF1（1～291aa）、EF2（292～402aa）和EF3（403-500aa），又将EF1分成4个相互重叠的小片段，分别是EF1-1（1～88aa）、EF1—2（68～155aa）、EF1—3（129～222aa）和EF1—4（202～291aa），将以上基因片段分别克隆到原核表达载体进行GST融合表达。SDS-PAGE电泳鉴定各融合蛋白均获得表达，各表达产物以包涵体的形式存在。通过Western blot分析表明融合蛋白GST-EF1、GST—EF2、GST—EF1—1、GST-EF1-2、GST—EF1—3和GST—EF1～4均能被阳性血清识别。通过ELISA鉴定，GST-EF2反应性最强，GST-EF1反应性比GST-EF2稍弱，而GST-EF3反应性最弱，GST-EF1-1、GST-EF1—2、GST-EF1—3和GST—EF1—4片段融合蛋白反应性与GST—EF1相似。由此本研究鉴定出1-402aa是E蛋白的抗原优势区域，在此区域内包括了12个Cys的保守区及域Ⅰ、域Ⅱ和域Ⅲ3个主要抗原域。E蛋白抗原优势区域的鉴定，为E蛋白抗原表位的鉴定以及针对于E蛋白诊断试剂的开发奠定了基础。

IDENTIFICATION OF MAJOR ANTIGENIC DOMAIN OF E PROTEIN OF JAPANESE ENCEPHALITIS VIRUS

Japanese encephalitis virus （JEV） is an arbovirus belonging to genus Flavivirus of family Flaviviridae and causes zoonotic disease in humans and animals. The envelope glycoprotein （E protein） of JEV is associated with viral attachment and fusion with host cells, determines the virus＇s hemagglutination ability, cellular tropism and viral virulence, and induces protective immune response. In order to map the immunological advantage domain of E protein, 3 pairs of primers were desi into 3 domains, EF1 （1-291 aa）, EF2 （292-402 aa） and EF3 （403-500 aa divided into 4 overlapping sub-domains, EF1-1 （1-88 aa）, EF1-2 （68-15 gned. The E protein was divided ）. The EF1 domain was further 5 aa）, EF1-3 （129-222 aa） and EF1-4 （202-291 aa）. These domains and sub domains were cloned into prokaryotic expression vector pGEX-6p-1, and expressed with GST tag. The fusion proteins were all visualized by SDS-PAGE analysis. The expressed products were in the form of inclusion bodies. Except EF3 domain, all other 2 domains and 4 sub-domains strongly reacted with positive sera in Western blot and ELISA, indicating that 1-402 aa of E protein was the immunological advantage domain. Results provide important information on studying the function of E protein and developing Japanese encephalitis diagnostic reagents.