鲤春血症病毒单克隆抗体建立及免疫学特性鉴定

Establishment of Hybridoma Cell Lines Secreting Anti-Spring Viraemia of Carp Virus Monoclonal Antibody and Identifi cation of Their Immunological Properties

制备高特异性的抗鲤春血症病毒单克隆抗体,并对其免疫学特性进行鉴定。采用差速离心法提纯的鲤春血症病毒作为免疫原,免疫Balb/c小鼠,4次免疫后,利用杂交瘤细胞技术进行细胞融合,经过多次亚克隆筛选出高敏感的特异性抗鲤春血症病毒单克隆抗体杂交瘤细胞株,体内诱生腹水制备抗鲤春血症病毒单克隆抗体并对其免疫学特性进行鉴定。融合后筛出两株杂交瘤细胞株9C11和1C12,亚型均为IgG2b型,κ链;间接ELISA测定腹水效价为104,与其他病毒株和细胞株不发生反应;western-blot结果显示单克隆抗体对应的抗原位点在相对分子质量(Mr)230 000的条带处;免疫氧化酶染色结果显示单克隆抗体能够特异地识别感染细胞内的鲤春血症病毒。本实验制备的抗鲤春血症病毒单克隆抗体特异性好,为鲤春血症病毒免疫学快速检测方法的建立奠定了基础。

The aim of the study is to generate high specificity monoclonal antibody against spring viraemia of carp virus （SVCV） and identify its immunological characteristics. The BALB/c mice were immunized with the SVCV purified by differential centrifugation. Spleen cells of immunized mice were collected and fused with SP2/0 myeloma cells. High sensitivity and high specificity monoclonal antibody was prepared after several times subcloning, and the immunological characteristics, characterized. Limited dilution method was used to subclone the positive clones. After three cycle of subcloning, two cell strains were obtained;it can secrete mAb against SVCV and were named as 9C11 and 1C12. The titer of mAbs ascites was 104 by the indirect ELISA. It belongs to IgG3 subclassκchain and couldn’t react with other fish viruses or fish cells lines in the ELISA test except SVCV. In the western blotting using mAbs （9C11 and 1C12）, a 230 kDa of protein band was observed when SVCV was used as antigen. The result of the immunoperoxidase test showed that the monoclonal antibody can specifically recognize SVCV in infected cells. The obtained mAb could be used to rapidly diagnosis and detect SVCV in aquaculture.