猪肌生成抑制素单克隆抗体的制备及鉴定

摘要

Balb /c mice were immunized with purified recombinant GST-MSTN protein.Hybridoma cell lines were set up by using hybridoma technique,and monoclonal antibodies were prepared.Using puri-fied C-termination function MSTN as detection antigen,titer of immuned mice serum antibody,titer of hy-bridoma culture supernatant,the type and immunoglobulin subclasses and additive index of antibody were determined with enzyme linked immunosorbent assay (ELISA).Western blot analysis was used as anti-bodies specific.The results showed that the experiment obtained 3 stable secreting anti-porcine myostatin monoclonal antibody cell line,named 3D3,1G8 and 10H1 .The titer of immuned mice serum antibody of the mice,detected by ELISA,was 1 ∶400,1 ∶800 and 1 ∶800.The ascites titer of 3 monoclonal antibody (3D3,1G8 and 10H1)were 1 ∶16 000,1 ∶32 000 and 1 ∶32 000.Western blot analysis showed that all of the monoclonal antibodies could specifically bind to recombinant MSTN protein.Immunoglobulin sub-classes of antibodies secreted by hybridoma cells displayed both in 3D3 are IgM,and 1G8 and 10H1 be-long to IgG2a.The results of three antibodies additive experiments show that 3D3,1G8 and 10H1 have different antigen recognition sites.%用纯化的重组 GST-MSTN 蛋白免疫 Balb/c 小鼠，采用杂交瘤技术制备鼠抗猪肌生成抑制素单克隆抗体。以纯化的 MSTN C 端成熟蛋白为检测抗原，采用 ELISA 的方法检测免疫小鼠血清抗体效价、杂交瘤细胞培养上清效价、纯化腹水效价、免疫球蛋白的类型和亚类，抗体相加指数。用 Western blot 检测抗体的特异性。结果表明：3D3、1G8和10H13株单克隆抗体细胞培养上清效价分别为1∶400，1∶800，1∶800，纯化腹水效价分别为1∶16000，1∶32000，1∶32000。Western blot 分析显示，3株单抗均能与重组 MSTN 蛋白特异结合。免疫球蛋白亚类鉴定显示杂交瘤细胞所分泌的抗体中3D3属于 IgM亚类，1G8和10H1属于 IgG2a 亚类。抗体相加试验结果表明3D3、1G8与10H1有不同的抗原识别位点。