尼帕病毒和亨得拉病毒核蛋白单克隆抗体的制备与鉴定

Preparation and characterization of monoclonal antibodies against recombinant nucleocapsid protein of Henipahvirus

尼帕病毒（Nipahvirus，NiV）和亨得拉病毒（Hendravirus，HeV）是近年来出现的2种新的高致病性副粘病毒，在我国尚未发现。为防范2种病毒在我国的出现，本研究开展了前瞻性工作，成功研制了针对2种病毒核蛋白（N）的单克隆抗体，可用于病毒监测与诊断。首先利用大肠杆菌表达的2种病毒N蛋白免疫BALB／c小鼠，细胞融合后应用间接免疫荧光的方法对杂交瘤细胞克隆进行筛选，获得了5株N蛋白特异单抗。单抗腹水的抗体效价均超过2×10^5，培养上清抗体效价1：64～1：256。Western—blot和间接免疫荧光试验证明，5株单抗均特异针对N蛋白，其中1株（H4D11）只与HeVN蛋白反应，而不与NiVN蛋白反应，表明它具有鉴别2种病毒的能力。所研制的单抗对建立2种病毒的检测技术，用于动物监测，以防范2种新发传染病在我国流行具有重要意义。

Henipahvirus contains Hendra virus （HeV） and Nipah virus （NiV）,are very dangerous agents caused of fetal Nipah and Hendra zoonoses ,which first emerged in 1998 in Malaysia and in 1994 in Australia respectively. These diseases may lead to the severe pathogenic damage on respiratory and central nervous system,usually associated with high morbidity and mortality in host animal and human. There is a need for rapid detection of the virus to determine etiological agent. In this paper,the recombinant nucleocapsid protein of Henipahvirus expressed in E. coli were used to immune BALB/c mouse,all hybridoma supernatants were selected by IFA using fixed sf9 insect cell expressed nucleocapsid protein of Henipahvirus ,finally,two hybridoma cell lines secreted against NiV N and three against HeV N were established,all the MAbs are IgG1 subtype except H2F5 ,which is IgM. The titers of hybridoma supernatants and ascites in I-ELISA are 1 ：64-1 ： 256 and 1 ： 204 800 respectively. The available dilution of all the MAbs employed in IFA are 1 ： 1 600-1 ： 3 200. Western-blot and IFA results show that four MAbs can crossreact with NiV N and HeV N,while H4D11 only react with HeV N. This means H4D11 only recognize the specific epitopes of HeVN ,it will benefit for diffenciation diagnosis.