SP-B中间产物特异性抗体的制备与鉴定

Preparation and Characterization of Polyclonal Antibodies against the Surfactant Protein B Intermediates

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 characterize the rabbit polyclonal antibodies against the surfactant protein （SP） B intermediates. Methods The human SP-B sequence was analyzed using Lasergene 7. 0 and Macvector 11.0. 4 software, and three epitopes were predicted. Three peptides were synthesized and conjugated to keyhole limpet hemocya- nin （KLH） via cysteine residue, which were used as immunogen to rabbits. The reactivity against the immunizing peptide of pre-immune and post-immune sera were screened by indirect enzyme-linked immunosorbent assay （ELISA）. The anti- sera were purified by immunoaffinity using specific antigen, then were concentrated by ultrafiltration. The purity of anti- bodies was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the reactivity of anti- bodies against the immunizing peptide was screened by indirect ELISA. The specificity of antibodies was characterized by western blot. Results The reactivity against the immunizing peptide of pre-immune sera was low, and that of post-im- mune was high enough. The antibodies had high purity and reactivity and excellent specificity. Conclusion Three poly- clonal antibodies against SP-B intermediates are prepared successfully, which provides a tool for further study of the pro- cessing of SP-B.