猪肌生成抑制素单克隆抗体的制备及鉴定  

Preparation and Identification of Porcine Myostatin Monoclonal Antibody

作　　者：车东升[1] 张春[1] 李占军[2] 李莉[2] 逄大欣[2] 欧阳红生[2] 

机构地区：[1]吉林农业大学动物生产及产品质量安全教育部重点实验室,长春130118 [2]吉林大学动物科学学院,长春130062

出　　处：《吉林农业大学学报》2013年第6期732-735,741,共5页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(31201763);吉林省教育厅青年基金项目(吉教科合字2009-362)

摘　　要：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MSTNC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10H1 3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∶16 000,1∶32 000,1∶32 000。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。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 immtmed 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, 1 G8 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.

关 键 词：猪 肌成生抑制素 单克隆抗体 

分 类 号：S828[农业科学—畜牧学] Q7[农业科学—畜牧兽医]