猪瘟病毒保护性抗原E2蛋白单抗的制备及其抗原表位的初步分析

Preparation and Epitope Analysis of Monoclonal Antibodies against CSFV Envelope Glycoprotein E2

应用大肠杆菌表达的猪瘟病毒主要保护性 E2抗原决定簇蛋白和全长 E2基因构建的 DNA疫苗免疫 BAL B/c小鼠。取免疫小鼠脾细胞与 SP2 /0骨髓瘤细胞进行融合 ,经克隆和间接 EL ISA筛选 ,获得了 A11、B2、E3、H6、D5、D86株稳定分泌抗猪瘟病毒 E2蛋白单克隆抗体 (Mc Ab)的杂交瘤细胞株。它们的腹水效价在 1∶ 80 0～ 1∶ 2 10 0 0 0之间。抗体类型鉴定结果表明 ,A11、E3、H6、D5和 D8为 Ig M类型 ,B2为 Ig G类型。随后用蛋白 A凝胶层析法和 PEG沉淀法分别纯化了 Ig G和 Ig M单抗。对纯化单抗进行的抗原识别表位研究结果初步表明 ,6株单抗可能识别 3种不同的E2抗原表位。

A panel of monoclonal antibodies(McAbs) against E2 protein of classical swine fever virus were generated by immunization of BALB/c with:a.E.coli-expressed one third part of N terminal of E2,which includes predominant neutralizing antigenic determinants,b.DNA vaccine based on entire E2 gene.All 6 hybridomas were characterized for their potency of secreting monoclonal antibody showing the antibody titers between 1∶800- 210 000 .The preliminary identification of antigenic epitopes recognized by those McAbs in ELISA by using synthetic peptides showed that these 6 McAbs could recognize three epitopes of immunogenic E2 protein.These antibodies will be used to delineate the relationship between E2 gene variation and the antigenic change of the virus.