日本脑炎病毒囊膜蛋白单克隆抗体的制备及特性分析  

Preparation and characterization of monoclonal antibodies against envelope protein of Japanese encephalitis virus

作　　者：倪慧[1] 王钰[1] 毛岩[1] 陈溥言[1] 杨梅[1] 曹瑞兵[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2016年第7期1-9,共9页Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)科研专项(201203082);南京农业大学国家级SRT项目(201510307053)

摘　　要：将日本脑炎病毒(JEV)囊膜蛋白Cap-ED3重组蛋白免疫BALB/c雌鼠,应用细胞融合技术制备单克隆抗体,经ELISA筛选获得4株稳定分泌识别JEV ED3蛋白单抗的杂交瘤细胞,命名为1B10、3F5、4H1和5F9。这4株单抗在Western blot和间接免疫荧光试验中均能特异识别JEV NJ2008株。制备4株单抗的小鼠腹水,5F9效价最高,为1∶409 600。空斑减少中和试验结果表明:4株单抗均没有中和活性。相加ELISA试验表明:4株单抗中3F5和4H1识别相同抗原表位,1B10、5F9分别识别其他的抗原表位,且差异较大。应用鸭坦布苏病毒(DTMUV)特异性抗原进行ELISA检测的结果表明:5F9识别的表位与DTMUV有交叉,其他3株识别的抗原表位与DTMUV无交叉。本研究获得了3株JEV囊膜蛋白特异性的单抗和1株识别JEV和DTMUV交叉抗原表位的单抗,为JEV抗原和抗体检测试剂盒的研制提供了良好的物质基础。Japanese encephalitis is a mosquito borne zoonosis caused by Japanese encephalitis virus（ JEV）,which threats both human and several kinds of livestock and has important impact in public health in China. In the present study,monoclonal antibodies（ MAbs） against JEV envelope protein were prepared by cell fusion technology. The purified cap and ED3 fusion protein which form viral like particle was used to immunize BALB / c female mice. For the screening of envelope specific MAbs,a soluble recombinant ED3 protein were expressed in E. coli BL21 cells. Analyzed by immunogical methods,MAbs 1B10 and 3F5,4h1 and 5F9 were selected on the basis of their reactivity. All of the four MAbs could recognize naive JEV NJ2008 strain in the western-blotting and IFA tests. The titers of antibodies secreted into the culture supernatant and ascitic fluid were measured by indirect ELISA and the highest antibody titer of ascitic fluid was 1 ∶ 409 600 for 5F9. However,none of the four MAbs was identified to have neutralizing activity against JEV NJ2008 strain in the plaque reduction neutralization test.The result of additional ELISA indicated that 3F5 and 4H1 recognized the same antigen epitope while 1B10 and 5F9 reacted with other antigen sites. Furthermore these MAbs had no cross reactivity with duck tembus virus（ DTMUV） except 5F9. The preparation of the four MAbs against JEV envelope protein provides a basis for development of ELISA kits for JEV antigen or antibody.

关 键 词：日本脑炎病毒 囊膜蛋白 单克隆抗体 鸭坦布苏病毒 交叉反应 

分 类 号：S852.6[农业科学—基础兽医学]