摘要
目的克隆日本乙型脑炎病毒（Japanese encephalitis virus，JEV）前膜蛋白（prM）编码基因，原核表达、纯化prM蛋白，以纯化产物为免疫原制备单克隆抗体（mAb）；方法从感染JEV病毒的鼠脑中克隆编码JEV prM蛋白的基因并将其克隆人原核表达载体pET32a，转化大肠埃希菌BL21（DE3）LysS后以IPTG诱导表达。表达产物经Ni-NTA纯化后进行SDS-PAGE分析。用纯化的蛋白免疫BALB／c小鼠，经细胞融合和亚克隆后筛选出能分泌识别prM蛋白的mAb的杂交瘤细胞株。用Western Blot和免疫组化方法检测单克隆抗体的特异性。结果从鼠脑中克隆出约500bp的JEV prM基因，将其克隆人原核表达载体中，在大肠埃希菌中获得了较好表达。纯化的prM蛋白免疫BALB／c小鼠，经传统细胞融合及筛选方法制备出单克隆抗体，抗体滴度为10^5。ELISA、Western Blot和免疫组化检测结果证实该株单抗具有较好的特异性。结论成功的表达和纯化了日本乙型脑炎病毒的prM蛋白，并完成了单克隆抗体的制备，为乙型脑炎病毒感染的早期诊断及预防的研究奠定了良好的基础。
Objective To prepare monoclonal antibody （mAb） against prM epitope.Methods The gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E. coli BL21 （DE3）LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 ceils. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay. Results mAb against prM epitope of JEV was prepared succesfully. Conclusion The obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2008年第1期65-67,共3页
Chinese Journal of Experimental and Clinical Virology
基金
基金项目：国家科技攻关项目（2003BA712A09-01）
关键词
脑炎病毒
日本
病毒包膜蛋白质类
原核细胞
表达的序列标记
抗体
单克隆
Encephalitis virus, Japanese
Viral envolope proteins
Proxaryotic cells
Expressed sequence tags
Antibodies, monoclonal
