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目的研制Ⅰ型登革病毒包膜蛋白Ⅲ区(dengue virus 1 envelope protein domainⅢ,DENV-1 EDⅢ)单克隆抗体,并鉴定其血清型特异性、相对表位和中和活性。方法采用DENV-1 EDⅢ重组蛋白为免疫原,免疫Balb/c小鼠,制备抗DENV-1 EDⅢ单抗,鉴定单抗的免疫学特性和交叉反应性,噬斑减少中和实验鉴定其中和活性,抗体与抗体间竞争抑制分析相对表位。结果共获得抗DENV-1 EDⅢ单抗28株,其Ig亚类鉴定24株为Ig G1,2株为Ig G2a,2株为Ig G2b,包含13株DENV-1血清型特异性单抗,9株DENV病毒特异性单抗,其它6株交叉反应性单抗以不同的方式同各型DENV EDⅢ反应,至少识别5个不同位点,其中27株抗体对不同血清型登革病毒表现出不同的中和活性。结论成功获得了针对DENV-1 EDⅢ的特异性单抗及交叉性中和单抗,将为进一步研究登革病毒EDⅢ蛋白的结构与功能、疫苗和治疗性抗体的研发奠定基础。
Objective To develop a monoclonal antibody against dengue virus 1 envelope protein domain Ⅲ (DENV-1 EDⅢ) and identify its serotype specific, relative epitope and neutralizing activity. Methods Immunization of Balb / c mice with DENV-1 EDⅢ recombinant protein was used to prepare anti-DENV-1 EDⅢ monoclonal antibody. The immunological and cross-reactivity of monoclonal antibodies against DENV-1 were identified. The plaque reduction neutralization assay was used to identify the neutralizing activity Antibodies compete with antibodies to inhibit relative epitope analysis. Results A total of 28 anti-DENV-1 EDⅢ monoclonal antibodies were obtained. Among them, 24 were Ig G1, 2 were Ig G2a and 2 were Ig G2b, including 13 DENV-1 serotype-specific monoclonal antibodies 9 Strain DENV virus-specific monoclonal antibody, the other six cross-reactive monoclonal antibodies in different ways with each type of DENV ED Ⅲ reaction, identify at least five different sites, of which 27 antibodies against different serotypes of Dengue virus showed different Neutralize activity. Conclusion The specific monoclonal antibodies against DENV-1 EDⅢ and the crossed neutralizing monoclonal antibodies were successfully obtained and laid the foundation for the further study on the structure and function of the EDⅢ protein of dengue virus and the development of vaccines and therapeutic antibodies.