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目的制备和鉴定表面活性物质蛋白B(surfactant protein B,SP-B)加工处理过程中部分中间产物的特异性抗体。方法利用Lasergene 7.0和Macvector 11.0.4软件进行抗原表位分析,确定作为抗原的3条多肽序列,将人工合成的多肽与钥孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)偶联,免疫兔制备抗血清,间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测免疫前血清背景和免疫后血清效价,抗原亲和纯化抗血清,超滤浓缩抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)检测抗体纯度,间接ELISA法检测抗体效价,蛋白质印迹(Western blot)鉴定抗体特异性。结果免疫前兔血清背景均低,免疫后抗血清效价均达到要求,抗原亲和纯化后所得抗体纯度及效价高,特异性好。结论成功制备了3种SP-B中间产物特异性多克隆抗体,为深入研究SP-B的加工处理奠定了基础。
Objective To prepare and identify specific antibodies of some intermediate products during the processing of surfactant protein B (SP-B). Methods Antigen epitope analysis was performed using Lasergene 7.0 and Macvector 11.0.4 software. Three polypeptide sequences were identified as antigens. Synthetic peptides were conjugated to keyhole limpet hemocyanin (KLH) The antiserum and the indirect enzyme-linked immunosorbent assay (ELISA) were used to detect the serum background before immunization and the serum titer after immunization. Antigen affinity purified antiserum, ultrafiltration concentrated antibody, sodium dodecyl sulfate-poly The purity of the antibody was detected by SDS-PAGE, the antibody titer was detected by indirect ELISA, and the specificity of the antibody was identified by Western blot. Results The serum of pre-immune rabbit serum was low, the antiserum titer reached the requirement after immunization, and the purity and titer of the purified antibody were high and the specificity was good. Conclusion Three SP-B specific polyclonal antibodies were successfully prepared, which laid the foundation for the further study on the processing of SP-B.