抗鸡传染性支气管炎病毒核蛋白单克隆抗体的制备及抗原表位鉴定

Preparation of monoclonal antibodies against the nucleocapsid of Infectious bronchitis virus and epitope identification

为鉴定鸡传染性支气管炎病毒(IBV)抗原表位,本研究将IBV tl/CH/LDT3/03株免疫BALB/c小鼠,将其脾细胞与SP2/0骨髓瘤细胞融合,经克隆筛选,得到两株针对IBV核(N)蛋白的单克隆抗体(MAb)6H3和6F9,其染色体数目分别为90条和102条,MAb亚类鉴定分别属于IgG1和IgG2b,轻链均为κ链。对MAb6H3和6F9的抗原表位进行初步鉴定,将N基因分成8个片段克隆于pGEX-6p-1载体中,转化BL21(DE3)内诱导表达。经western blot分析,MAb6H3表位的初步定位在aa80～aa99,而MAb6F9表位的初步定位在aa64～aa82。而且MAb6F9还能特异性识别其他5株IBV的天然N蛋白,推测它们含有一个相同的B细胞表位。

BALB/c mice were immunized with intact virion of infectious bronchitis virus （IBV） and the spleen was fused with SP2/0 myeloma cells. Two MAbs designated 6H3 and 6F9 against the nucleocapsid of IBV were obtained. The number of chromosome of hybridomas 6H3 and 6F9 were 90 and 102,respectively. In order to map epitopes of two MAbs,eight truncated N gene fragments were cloned into the pGEX-6p-1 and transformed to E. coli BL21 （DE3）. Western blot analysis showed that MAb 6H3 and MAb 6F9 recognized linear epitopes located at aa 80 to aa 99 and aa 64 to aa 82,respectively. Moreover,MAb 6F9 could recognize the native nucleocapsid of other four different IBV strains,indicating that the five different IBV strains shared one B cell linear epitope.