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根据GenBank公布的日本脑炎病毒(Japanese Encephalitis Virus, JEV) SA14 14 2株的核酸序列和人流感病毒的血凝素基因(ha)序列, 设计一对特异性引物, 用 PCR方法扩增编码 JEV囊膜蛋白主要抗原域基因, 其中含ha基因主要核苷酸序列。将PCR产物定向克隆入原核表达载体 pET 32a(+), 构建原核表达载体 pET EHA。阳性质粒转化BL21(DE3)宿主菌, 经 IPTG诱导获得表达, 重组蛋白以包涵体的形式存在。Western blot分析表明表达产物具有良好的免疫学活性。利用纯化的表达产物与流感病毒血凝素单抗及乳胶建立了诊断日本脑炎病毒抗体水平的乳胶凝集试验。结果表明乳胶凝集方法具有简便快速、敏感性高、特异性强、价格低廉、可现场检测等优点, 是一种适合基层兽医单位用于日本脑炎病毒抗体水平检测的新方法。
A pair of specific primers was designed based on the nucleotide sequence of Japanese Encephalitis Virus (JEV) strain SA14142 published in GenBank and the hemagglutinin gene (ha) sequence of human influenza virus. A pair of specific primers were designed and used to amplify the JEV Envelope protein major antigen domain gene, which contains ha gene major nucleotide sequence. The PCR product was cloned into the prokaryotic expression vector pET 32a (+) and the prokaryotic expression vector pET EHA was constructed. Positive plasmids were transformed into BL21 (DE3) host strain and expressed by induction of IPTG. The recombinant protein was in the form of inclusion bodies. Western blot analysis showed that the expressed product has good immunological activity. A latex agglutination test for the diagnosis of Japanese encephalitis virus antibody levels was established using purified expression products with influenza virus hemagglutinin and latex. The results showed that the latex agglutination method is a new method suitable for the detection of Japanese encephalitis virus antibodies by the veterinary unit at the grass-roots level because of its advantages such as simple and rapid, high sensitivity, high specificity, low cost and field detection.