RECOMBINANT DENGUE VIRUS ANTIGEN PRODUCTION

登革病毒(Dengue Virus，DEN)是一种引起登革热的黄病毒，是一种急性发热性疾病，在全球热带和亚热带地区发病率越来越高。这4个DEN血清型和其他黄病毒通常在流行地区共循环，加上它们广泛的抗原交叉反应，混淆了血清诊断。由于以前接触不同的DEN血清型可能导致严重后果(DHF/DSS)，因此DEN血清型识别对于指导患者的治疗策略和预测DHF/DSS暴发很重要。商业上可用的诊断测试依赖于仅针对黄病毒的完整病毒抗原。将利用编码截短的DEN包膜蛋白与大肠杆菌MBP融合的质粒，开发用于商业生产针对每个DEN血清型的重组DEN抗原(RDEN)的方法。实验室规模的纯化工作将确定产生未聚集重组蛋白的亲和层析条件；随后将进行高达30升的生产批次。抗原的特异性和亲和力将通过免疫印迹、斑点EIA和微孔板ELISA法与人血清和小鼠抗体进行评估，这些抗体代表了IFA、ELISA法和中和试验确定的一系列特异性和效价。将启动稳定性测试。将RDEN纳入我们的商业酶联免疫吸附试验将提供所需的特异性，并避免繁琐和危险的病毒生产。拟议的商业应用：登革热是一种主要的公共卫生威胁，估计每年有超过1亿例病例，而且其地理范围正在扩大。被广泛用作诊断辅助手段的血清学检测依赖于相对非特异性的整个病毒抗原。建议用重组抗原取代这些抗原，这将通过提高特异性显著提高对登革热的检测。PanBio indx已经在40多个国家和地区销售登革热ELISA试剂盒，市场预计还会扩大。改进后的酶联免疫吸附试验应能被广泛接受。

Dengue virus (DEN) is a flavivirus causing dengue fever, an acute febrile illness which occurs with increasingly high incidence in the tropics and subtropics worldwide. The 4 DEN serotypes and other flaviviruses commonly cocirculate in endemic regions, which together with their extensive antigenic cross reactivity confound serodiagnosis. Because severe outcomes (DHF/DSS) may result from previous exposure to a different DEN serotype, DEN serotype identification is important in indicating patient treatment strategies and in predicting DHF/DSS outbreaks. Commercially available diagnostic tests rely on whole virus antigens which are only flavivirus-specific. Methods will be developed for commercial production of recombinant DEN antigens (rDEN) specific for each DEN serotype using plasmids encoding truncated DEN envelope proteins fused with E. coli MBP. Bench-scale purification efforts will define affinity chromatography conditions which yield unaggregated recombinant proteins; scale up to 30-liter production lots will follow. Antigens will be evaluated for specificity and avidity by Western blotting, dot EIA, and microplate ELISA with human sera and mouse antibodies representing a range of specificities and titers determined by IFA, ELISA, and neutralization tests. Stability tests will be initiated. Incorporation of rDEN into our commercial ELISA tests will provide needed specificity and avoid laborious and hazardous virus production. PROPOSED COMMERCIAL APPLICATIONS: Dengue is a major public health threat, with over 100 million cases estimated per year, and its geographic range is expanding. Serologic tests, widely used as diagnostic aids, rely on relatively nonspecific whole virus antigens. Replacement of these with recombinant antigens is proposed, this will significantly enhance detection of dengue by contributing specificity. PanBio InDx already distributes dengue ELISA kits in over 40 countries and the market is expected to expand. The improved ELISA tests should receive wide acceptability.