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目的以Ⅰ型登革病毒pr M蛋白为靶抗原,制备相应的多克隆和单克隆抗体,并进行初步鉴定。方法 RTPCR扩增Ⅰ型登革病毒全长pr M基因,转入克隆载体和构建重组表达载体,以原核表达及纯化后的pr M蛋白作为免疫原分别免疫新西兰大白兔和BALB/c小鼠,采集免疫兔血清,制备抗pr M蛋白多克隆抗体;取小鼠脾细胞与骨髓瘤细胞融合,经HAT选择培养、间接ELISA筛选阳性克隆,获得特异性单克隆抗体的杂交瘤细胞株,制备抗pr M蛋白单克隆抗体,应用亲和层析法纯化抗体,Western-blot鉴定抗体特异性,间接ELISA检测抗体效价。结果获得全长pr M的多克隆抗体及1株能稳定分泌抗pr M蛋白的单克隆抗体杂交瘤细胞株,抗体的效价高,特异性好。结论制备了Ⅰ型登革病毒pr M多克隆抗体和1株单克隆抗体,为进一步探讨登革病毒pr M抗体依赖的感染增强作用奠定基础。
Objective To construct the corresponding polyclonal and monoclonal antibodies against prM protein of dengue virus type Ⅰ and to identify them. METHODS: The full-length pr M gene of dengue type Ⅰ was amplified by RT-PCR and cloned into the cloning vector and the recombinant expression vector. The prM protein was purified and used as immunogen to immunize New Zealand white rabbits and BALB / c mice, Immunized rabbit serum was collected to prepare anti-pr M protein polyclonal antibody. The mouse spleen cells were fused with myeloma cells and selected by HAT. The positive clones were screened by indirect ELISA to obtain specific monoclonal antibody hybridoma cell lines, pr M protein monoclonal antibody, affinity chromatography purified antibodies, Western-blot identification of antibody specificity, indirect ELISA detection of antibody titer. RESULTS: A polyclonal antibody with full length pr M and a monoclonal antibody hybridoma cell strain that can stably secrete anti-pr M protein were obtained. The antibody titer is high and the specificity is good. Conclusion The polyclonal antibody to prM Ⅰ of dengue virus and one monoclonal antibody were prepared, which laid the foundation for further study on the enhancement of prM-dependent infection by dengue virus.