鸡肺表面活性蛋白A的表达及其多克隆抗体的制备  

Expression of chicken pulmonary surfactant protein A and preparation of its polyclonal antibodiy

作　　者：刘红梅[1] 王浩[1] 邵颖[1] 黄博言 周秀红[1] 潘玲[1] 王桂军[1] 祁克宗[1] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《细胞与分子免疫学杂志》2014年第1期63-65,70,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：安徽省教育厅自然科学基金重点项目(KJ2012A112)

摘　　要：目的表达鸡肺表面活性蛋白A(cSP-A)重组蛋白,并制备鼠抗cSP-A多克隆抗体。方法人工合成cSP-A的凝集素糖识别区(CRD)基因(cSP-A-CRD),亚克隆到pGEX-6P-1载体中进行原核表达,对表达产物进行鉴定。切胶纯化法纯化重组蛋白,并免疫小鼠制备抗cSP-A多克隆抗体,ELISA检测抗体效价,Western blot法、免疫荧光组织化学染色和ELISA鉴定该抗体的特异性和适用范围。结果获得鼠抗cSP-A血清,效价达到105;Western blot法结果表明多抗血清具有良好的特异性和反应性,并可用免疫荧光组织化学技术检测鸡肺上皮细胞表达的cSP-A和用间接ELISA检测鸡肺灌洗液中存在的水溶性cSP-A蛋白。结论成功表达了cSP-A重组蛋白并制备了小鼠抗cSP-A多克隆抗体。Objective To express chicken pulmonary surfactant protein A （cSP-A） in Escherichia coli and prepare polyclonal antibodies against the recombinant cSP-A. Methods The carbohydrate recognition domain （CRD） gene of cSP-A （cSP-A-CRD） was artificially synthesized, subcloned into the prokaryotic expression vector pGEX-6P-1, and expressed in Escherichia coli BL21 （ DE3 ）. After the expression products were identified by SDS-PAGE, the gel containing the target protein was cut and further purified. Mice were innoculated with the purified recombinant protein to prepare polyclonal antibodies againt cSP-A. The antibody titer was determined by ELISA, and the specificity and application scope of the polyclonal antibodies were analyzed by Western blotting, immunohistochemical fluorescence assay and ELISA. Results The serum against cSP-A was obtained and had an ELISA titer of 1 x 105. The serum also showed excellent specificity and reactivity as demonstrated by the Western blotting. It could be used to detect the cSP-A in chicken lung epithelial cells by immunohistochemical fluorescence staining and could also be used to detect the hydrosoluble cSP-A in chicken bronchoalveolar lavage fluid by indirect ELISA. Condusion The recombinant cSP-A was successfully expressed, and its mouse polyclonal antibodies were obtained.

关 键 词：鸡肺表面活性蛋白A 糖识别区 原核表达 多抗 

分 类 号：R392.11[医药卫生—免疫学] S831.99[医药卫生—基础医学]