Ⅱ型登革病毒非结构蛋白1多克隆抗体制备和免疫特征分析

Preparation and immune signature analysis of anti-dengue virus type 2 nonstructural protein 1 in vitro

目的制备抗Ⅱ型登革病毒(dengue virus type 2,DENV2)非结构蛋白1(nonstructural protein 1,NS1)多克隆抗体,分析该抗体的免疫特性。方法采用NS1蛋白免疫BALB/c小鼠,取血分离血清,纯化获得抗NS1抗体。酶联免疫吸附试验法(enzyme linked immunosorbent assay,ELISA)分析抗NS1抗体与NS1蛋白和DENV2结合特性。免疫荧光法测定抗NS1抗体与人微血管内皮细胞株1(human microvascular endothelial cell 1,HMEC-1)的交叉反应性。ELISA法分析抗NS1抗体、HMEC-1和新鲜同系小鼠血清共培养体系中活化补体的含量。结果抗NS1抗体能与NS1和DENV2特异性结合,抗体最高滴度分别为1∶1 280和1∶640。抗NS1抗体能与HMEC-1细胞交叉结合,抗NS1抗体、HMEC-1和新鲜同系小鼠血清共培养上清液中C3a、C4a、C5a和s C5b-9含量均显著高于HMEC-1和新鲜同系小鼠血清共培养(均P<0.05),以及PBS、HMEC-1和新鲜同系小鼠血清共培养(均P<0.05)。结论抗DENV2 NS1抗体能交叉结合于血管内皮细胞并激活补体系统,可能在登革出血热的免疫病理机制中起重要作用。

Objective To prepare the polyclonal antibodies directed against dengue virus type 2（DENV2） nonstructural protein 1（NS1）（anti-NS1 antibodies） and to analyze the immunological characteristics of the antibodies. Methods High titer polyclonal anti-NS1 antibodies were obtained from BALB/c mice immunized with DENV2 NS1. The binding ability of anti- NS1 antibodies to NS1 protein or DENV2 was detected by enzyme linked immunosorbent assay（ELISA）. The cross-reactivity of the antibodies with human microvascular endothelial cell 1（HMEC-1） was demonstrated using immunofluorescence assay. The levels of activated complement in the co- culture systems of anti- NS1, HMEC- 1 and fresh syngenic mice sera were measured using ELISA. Results Anti-NS1 antibodies could bind to NS1 and DENV2. The highest titer ofthe antibodies was 1：1 280 and 1：640 respectively. Anti-NS1 antibodies could cross bind to HMEC- 1. The levels of C3 a, C4 a, C5 a and s C5b-9 in the co-culture systems of anti-NS1, HMEC-1 and fresh syngenic mice sera were sharply higher than those in the co-culture systems of HMEC-1 and fresh syngenic mice sera（P〈0.05） and the co- culture systems of PBS, HMEC- 1 and fresh syngenic mice sera（P〈0.05）. Conclusion These results indicate that anti- NS1 antibodies can crosslink to vascular endothelial cell and activate the complement system, which may be involved in the immunopathologic mechanism of dengue hemorrhagic fever.