口蹄疫病毒非结构蛋白3B单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibody (MAb) against Non-structural Protein 3B of Foot-and-mouth Disease

用E.coli原核表达并纯化的口蹄疫病毒(Foot-and-mouth disease virus,FMDV)非结构蛋白3B免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,间接ELISA筛选出分泌鼠IgG的杂交瘤细胞株,将该杂交瘤细胞注射小鼠产生的腹水,用间接ELISA法筛选获得6株能稳定分泌抗3B蛋白单克隆抗体的杂交瘤细胞株,分别命名为3E5、4B1、4D7、4E11、7B2、8B11。鉴定结果显示,4B1和4E11细胞分泌IgG1,其余4株细胞分泌IgG2b;纯化后6株腹水单抗的纯度达90%以上,对3B蛋白的ELISA滴度均可达到1∶100000以上;6株单抗均不与FMDV结构蛋白VP1和3D非结构蛋白发生反应;间接免疫荧光试验证明所制备的单抗能够识别3B蛋白;杂交瘤细胞株连续培养3个月以及冻存6个月后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。

The monoclonal antibodies against protein 3B of foot-and-mouth disease virus were generated by fusing spleen cells from 3B immunized BALB/c mouse with SP2/0 mouse myeloma cells. The culture of hybridoma was screened by I-ELISA for detection of specific mouse IgG against FMDV 3B protein. Six strains of hybridoma which secreted monoclonal antibodies against protein 3B were determined and named 3E5, 4B1, 4D7, 4E11 , 7B2 and 8B11. The isotypes were identified to be IgG1 and IgG2b. The titer of these MAbs were over 10^5 by indirect EHSA. The purities of purified MAbs in ascites were more than 90%. Furthermore, the MAbs showed high specificities to protein 3B and had low reactivities to structural proteins and other non-structural protein. The MAbs showed strong reactivities in IFA for detection of the FMDV infected BHK-21cell. These hybridoma cells could be cultured for continuous three months and kept a stable condition.