Ⅰ型登革病毒prM抗体的制备及初步鉴定  

Preparation and primary identification of polyclonal antibodies and a monoclonal antibody against dengue virus type 1 prM

作　　者：王裴[1] 张宝[1] 谢茜[1] 余健海 何晓恩 赵卫[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院三级生物安全实验室,广东广州510515

出　　处：《中国卫生检验杂志》2015年第11期1689-1691,1695,共4页Chinese Journal of Health Laboratory Technology

基　　金：NSFC-广东联合基金(U1132002);国家自然科学基金(31270974;31470271)

摘　　要：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 prepare and primarily identify polyclonal antibodies and a monoclonal antibody against dengue virus type 1 （ DENV1 ） prM protein. Methods The whole sequence of DENV 1 prM were amplified with RT - PCR and inserted into cloning vector and prokaryotic expression vector. New Zealand white rabbits and BALB/C mice were immunized with purified prM protein to prepare polyclonal antibodies. The spleen cells of immunized mice were fused with myeloma cells and cultured on HAT Media Supplement. A hybridoma cell line was selected by indect ELISA. The monoclonal antibody and polyclonal antibodies were respectively purified from ascites and serum by affinity chromatography. The specificity and sensitivity of the antibody were determined by ELISA and Western - blot after purification. Results Polyclonal antibodies and a monoclonal antibody against prM with high antibody titers and specificity were obtained. Conclusion The productions of DENV1 anti - prM lay a good foundation to perform further studies on mechanism for antibody - dependent enhancement.

关 键 词：登革病毒 多克隆抗体 单克隆抗体 抗体依赖的感染增强作用 

分 类 号：R392.11[医药卫生—免疫学]