鸡传染性支气管炎病毒S1蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies against S1 protein of avian infectious bronchitis virus

目的制备鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)S1蛋白单克隆抗体,并进行鉴定。方法用差速离心纯化的IBV四川分离株Sczy3免疫BALB/c小鼠,取脾细胞与骨髓瘤细胞SP2/0融合。利用IBV鼠高免血清建立间接ELISA,筛选杂交瘤细胞。强阳性孔经有限稀释法亚克隆,获得分泌单克隆抗体的杂交瘤细胞株,并制备腹水。用鼠单克隆抗体亚型鉴定试剂盒鉴定单抗的亚型,Western blot法分析单抗的反应原性,ELISA法分析单抗的特异性及其与不同IBV毒株的交叉反应性。结果经连续4次亚克隆,获得2株能稳定传代并分泌抗IBV Sczy3株单克隆抗体的杂交瘤细胞株1C8C1和2C10E7,其腹水效价分别为1∶12 800和1∶25 600。2株杂交瘤细胞分泌的单抗均为IgM亚型;均可特异性识别S1蛋白;2株单抗只与IBV发生特异性反应,与新城疫病毒(Newcastle disease virus,NDV)、禽流感病毒(avian influenza virus,AIV)H9亚型和AIV H5抗原均不反应,与其他5种基因型的10株IBV毒株均可产生交叉反应。结论成功制备了2株IBV S1蛋白单克隆抗体,为IBV的检测、抗原表位筛选及致病机理的研究等提供了材料。

Objective To prepare and identify the monoclonal antibodies （McAbs） against S1 protein of avian infectious bronchitis virus （IBV）. Methods BALB/c mice were immunized with IBV Sczy3 strain isolated from Sichuan Province, China and purified by differential centrifugation, of which the spleen cells were fused with myeloma SP2/0 cells. Indi- rect ELISA method was developed with mouse hyperimmune serum against IBV, by which strong positive cells were screened and subcloned by limiting dilution method to obtain the hybridoma cell strain and prepare the McAbs in ascites. The prepared McAbs were identified for subtype by mouse monoclonal antibody subtype identification kit, for reactogenic- ity by Western blot, and for specificity and cross reactivity with various IBV strains by ELISA. Results Two hybridoma cell strains secreting McAbs against IBV, which might be subcuhured stably, were obtained by subcloning for 4 times and named as 1C8C1 and 2C10E7, of which the titers in ascites were 1 ： 12 800 and 1 ： 25 600 respectively. Both the McAbs secreted were IgM, which recognized S1 protein specifically. The McAbs showed only specific reaction with IBV but no reactions with Newcastle disease virus （NDV）, avian influenza virus （AIV） subtype H9 or H5. However, the McAbs showed cross reactions with 10 IBV strains of other five genotypes. Conclusion Two McAbs against S1 protein of IBV were successfully prepared, which provided a material for the detection, screening of antigenic site and study on pathogenic mechanism of IBV.