伪狂犬病病毒gB和gE蛋白单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against gB and gE proteins of pseudorabies virus

采用大肠杆菌表达系统表达获得了伪狂犬病病毒(PRV)超强毒株ZJ01的gB和gE重组蛋白,用其免疫BALB/c小鼠,通过细胞融合、克隆和间接ELISA筛选,获得了4株能稳定分泌抗PRV单克隆抗体的杂交瘤细胞,细胞培养上清的ELISA抗体效价在1∶1 600~1∶6 400之间,腹水抗体效价达1∶4×105;连续培养20代,抗体效价基本一致。抗gB单克隆抗体B1B6和B3D7属于IgG2b,抗gE单克隆抗体E1B11和E5C10属于IgG1,轻链均为κ型。Western-blot和IFA结果显示,4株单克隆抗体均能与PRV流行毒株ZJ01、HZ、SD、NJ、LA发生特异性反应,B1B6和B3D7还能与PRV gE基因缺失疫苗毒株Bartha-K61发生反应,但E1B11和E5C10不能与Bartha-K61发生反应。

The recombinant gB and gE proteins of the very virulent strain ZJ01 of pseudorabies virus(PRV)were obtained by the E.coli expression system,which were used to immune BALB/c mice.Four monoclonal antibodies(McAbs)were prepared by cell fusion,cloning and indirect enzyme-linked immunosorbent assay(ELISA).The ELISA titers in supernatant were from 1∶1 600 to 1∶6 400,and the titers in ascites were more than 1∶4×105.The B1B6 and B3D7McAbs against gB protein both belong to IgG2 b subtype withκchain,and E1B11 and E5C10McAbs against gE protein both belong to IgG1 subtype withκchain.Western-blot and IFA results showed that the four McAbs could all reacted specifically with PRV wild strains,ZJ01,HZ,NJ,SD and LA.While B1B6 and B3D7hybridoma cell lines not only reacted with PRV wild strains,but also reacted with PRVgE-strain Bartha-K61.However,E1B11 and E5C10could not reacted with PRVgE-strain Bartha-K61.The results indicated that the preparation of PRV monoclonal antibodies could provide valuable tools for further development of antigen epitope analysis and specific diagnosis reagent.