抗登革病毒2型E蛋白单克隆抗体的制备和生物学特性的鉴定  

Preparation and Characterization of Monoclonal Antibody Against DEN Envelope Pretein

作　　者：陶萍[1] 帅光泽[2] 赖梅梅[1] 王雪梅[1] 郑健[1] 黄爱龙[1] 

机构地区：[1]重庆医科大学附属第二医院病毒性肝炎研究所感染性疾病分子生物学教育部重点实验室,重庆400016 [2]成都大学校医院,成都610106

出　　处：《生物技术通报》2008年第5期145-148,共4页Biotechnology Bulletin

摘　　要：为了制备可用于胶体金快速检测试纸条的抗登革病毒2型(DEN2)E蛋白单克隆抗体(mAb),通过基因克隆获得E基因与质粒pET32a(+)的重组质粒,将重组质粒转化入大肠杆菌BL21,IPGT诱导表达重组蛋白。以DEN2重组E蛋白免疫BALB/c小鼠,采用杂交瘤技术制备抗DEN2E蛋白的mAb,以间接ELISA法和Western blot进行mAb特异性鉴定;同时采用间接ELISA法鉴定mAb的Ig亚类。结果表明获得1株可分泌特异性mAb的杂交瘤细胞(7C7),其抗体亚类为IgG1。Western blot显示该株mAb能特异识别重组pET32a-DEN2E蛋白。因此,成功制备出抗DEN2E的1株mAb,为建立快速特异检测登革病毒感染的实验方法提供了有力的工具。To prepare monoclonal antibody（mAb）against DEN Envelope Pretein. The gene of dengue virus type 2 （DEN2）Envelope Pretein strain was inserted into cloning vector pET-32a.Then it was linked to expression vector pET- 32a （＋）and transfected to E.eoli BL21 cells. The E gene from DEN2 was expressed by IPTG induction. BALB/c mice were immunized by recombinant DEN2 E protein. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line SP2/0 cells. The hy-Bridoma cells that secreted anti-DEN2 E protein mAbs were cloned by limited dilution method. The specificity of mAb was tested by ELISA and Western blot analysis.The subtype of mAb was tested by ELISA. Results showed that from over 124 positive hybridomas which secreted anti-DEN2 E protein mAbs,one hybridoma was screened out,designated 7C7,and chromosome analysis revealed that the obtained hybridoma featured universal characteristics of the monodonal hybridoma cells which secreted mAb. The subtype of 7C7 was IgG1. Thus one specific mAb against DEN2 E protein was prepared successfully and identified primarily.

关 键 词：登革病毒 单克隆抗体 生物学特性 E蛋白 

分 类 号：Q813[生物学—生物工程]