登革病毒2型prM蛋白的表达及其抗体的ADE作用研究  

Expression of dengue virus type 2 prM protein and analysis of the antibody-dependent enhancement effect of its polyclonal antibody

作　　者：冯俊杰[1,2] 罗雅艳[1,2] 周俊梅[1,2] 方丹云[1,2] 曾谷城[1,2] 晏辉钧[1,2] 江丽芳[1,2] 

机构地区：[1]中山大学中山医学院微生物学教研室,广东广州510080 [2]中山大学热带病防治研究教育部重点实验室,广东广州510080

出　　处：《热带医学杂志》2013年第4期377-381,411,共6页Journal of Tropical Medicine

基　　金：国家自然科学-广东省联合基金(U1132002;U0632002);国家自然科学基金(31270974);国家(地区)合作与交流项目(81261160323)

摘　　要：目的建立登革病毒2型(DENV-2)前膜蛋白(prM)的原核稳定表达系统,并制备prM多克隆抗体,研究登革病毒特异性prM抗体的中和作用及抗体依赖性感染增强作用(ADE)。方法应用大肠杆菌表达系统表达全长DENV-2 prM蛋白,经电洗脱法纯化重组蛋白,免疫家兔,制备兔抗prM蛋白多抗;应用空斑减数中和试验(PRNT)和流式细胞术(FCM)分别检测prM兔多克隆抗体的中和作用和ADE效应。结果重组prM蛋白在原核系统可获得高效表达,prM蛋白的表达量占大肠杆菌全菌蛋白的15%左右;其免疫血清经Western blotting法证实为登革病毒特异性prM多克隆抗体,用ELISA法检测效价为1:800 000。PRNT表明,prM抗体对成熟和不成熟DENV-2感染C6/36细胞中和作用有限,最大中和程度分别为49.84%和50.22%;FCM结果表明,prM抗体能在较大的抗体稀释度范围(10-2～10-6)增强成熟和不成熟DENV-2感染,且在10-3稀释度时达到最高峰。结论重组DENV-2 prM蛋白有很强的免疫原性,其诱生的prM抗体对登革病毒仅有较弱的中和活性,但却呈现出较强的ADE作用,揭示prM抗体是一种感染增强性抗体。Objective To establish a stable expression system of DENV-2 prM protein and study the neutralizing effects versus antibody-dependent enhancement （ADE） activity of its polyclonal antibody （pAb） towards standard DENV-2 and immature DENV-2. Methods The DENV-2 prM protein was expressed in E.coli and then purified by electroelution. The purified protein was used to immunize the rabbit to produce pAb directed to prM. The neutralizing effects versus ADE activities of pAb against prM were tested by PRNT and FCM, respectively. Results The prokaryotic system had successfully expressed the recombinant protein with a high level of expression. The specificity of the polyclonal antibodies was verified by Western blotting and the titer of the polyclonal antibodies was 1：800 000. The result of PRNT suggested that the pAb against prM could not completely neutralize the infectivity of standard DENV-2 and immature DENV-2 even at a high concentration. The results of FCM suggested that the polyclonal antibodies were able to enhance the infection of standard DENV-2 and immature DENV-2 over a ＇wide range of concentrations （10-2-10-6）. Conclusions The recombinant prM protein had a strong immunogenicity. The pAb to prM showed a weak neutralizing activity towards standard DENV-2 and immature DENV-2 but potently promoted ADE infection. These findings suggested that anti-prM pAb had an infection-enhancing effect.

关 键 词：登革病毒2型 prM蛋白 prM多克隆抗体 中和作用 抗体依赖性感染增强作用(ADE) 

分 类 号：R373.33[医药卫生—病原生物学]