猪链球菌2型次黄嘌呤核苷酸脱氢酶单克隆抗体的制备及其识别表位分析

Production and Epitope Studying of Monoclonal Antibody Against IMPDH in SS2

摘要:

用纯化的硫氧还蛋白-IMPDH融合蛋白免疫BALB/c小鼠, 取其脾细胞与SP2/0骨髓瘤细胞融合, 对杂交瘤细胞及时筛选, 阳性孔经4次有限稀释法克隆, 成功获得1A8、1F2、2D2和2D12共4株能稳定传代并分泌抗IMPDH的单克隆抗体(McAb)的杂交瘤细胞株。4株腹水型单克隆抗体间接ELISA效价分别为100×211、100×211、100×210和100×28。经Western-blot分析表明, 4株单抗与硫氧还蛋白-IMPDH融合蛋白均具有特异性反应, 并且通过4种IMPDH全基因分片段缺失表达的融合蛋白, 分析了4株单抗所识别抗原决定簇的差异性, 发现1A8、1F2、2D2识别表位的编码基因集中在IMPDH基因片段的627 bp~790 bp之间, 2D12识别表位的编码基因则集中在IMPDH基因片段的411 bp~790 bp之间。猪链球菌2型中IMPDH单克隆抗体的获得及相应表位分析为研究IMPDH蛋白的生物学活性及免疫学活性奠定了基础。

Abstract:

Fusion protein Thioredoxin (Trx A)–IMPDH of Streptococcu suis type 2 (SS2) was purified with affinity chromatograph. Spleen cells from BALB/c mice which were immunized with purified fusion protein were fused with SP2/0 myeloma cells in order to product monoclonal antibody against IMPDH. Four hybridoma cell strains against IMPDH (named 1A8, 1F2, 2D2 and 2D12 respectively) were developed after four times of limiting dilution assay. The indirect ELISA titers of their ascites were 100×211, 100×211, 100×210, 100×28. The results of Western-blot indicated that the four McAbs only reacted with Trx A–IMPDH but not reacted with Trx A, that showed their good specificity. Meanwhile, the difference of their recognizing epitopes was analysed by means of four proteins which were respectively producted by deleting specific genes. The results showed that 1A8, 1F2 and 2D2’s corresponding epitopes are in 627 bp~790 bp sites of IMPDH gene and 2D12’s is in 411 bp~790 bp sites. The successful preparation of monoclonal antibodies against IMPDH of SS2 and the analysis of epitopes would facilitate the further research on the biological and immunological activity of IMPDH.