牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies against E2 protein of Bovine viral diarrhea virus

为制备牛病毒性腹泻病毒(BVDV)的特异性单克隆抗体(MAb),用含有BVDV E2基因的真核表达质粒pcDNA-E2免疫4周龄～6周龄BALB/c鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,以BVDV作为抗原建立间接ELISA筛选及4次连续克隆获得3株能稳定分泌抗E2MAb的杂交瘤细胞系,分别命名为2E2、3D12、4D6。MAb亚类鉴定表明,2E2和4D6为IgM类,3D12为IgG2a亚类,轻链均为κ链。经western blot和间接免疫荧光检测证明3株MAb针对E2的构象表位,并且3D12株MAb能与BVDV及猪瘟病毒(CSFV)结合,2E2和4D6MAb只能与BVDV结合。

In order to prepare monoclonal antibodies（MAbs） against Bovine viral diarrhea virus/mucosal disease virus（BVDV）,BALB/c mice were immunized with pcDNA-E2,and the mouse splenic cells fused with SP2/0 cells.Hybridoma cells were screened by ELISA using BVDV as coating antigen,clone purified and characterized.Three hybridoma cells stably secreting anti-E2 MAbs 2E2,3D12,4D6,respectively were obtained.Identification of immunoglobulin type of MAbs showed that 2E2 and 4D6 were IgM type with κchain,3D12 was IgG2a subtype with κchain.Western blot and indirect immunofluorescence indicated that all MAbs were specific to the E2 conformational epitopes.The 3D12 MAb was able to react with BVDV and CSFV.However,2E2 and 4D6 could only react with BVDV.