摘要
目的:表达并纯化小鼠白细胞相关免疫球蛋白样受体-1(mLAIR-1)胞膜外区与人IgGFc段的融合蛋白,制备兔抗mLAIR-1胞膜外区的抗体。方法:构建真核表达载体,表达并纯化mLAIR-1-Fc融合蛋白。以mLAIR-1-Fc融合蛋白免疫家兔,用偶联mLAIR-1-Fc融合蛋白的Sepharose-4B亲和层析柱纯化多克隆抗体。用间接免疫荧光染色和流式细胞术鉴定多克隆抗体的特异性。结果:表达并纯化了mLAIR-1-hIgFc融合蛋白。以其免疫家兔,用亲和层析的方法纯化得到兔抗mLAIR-1胞膜外区的抗体,能与转染细胞和细胞表面天然mLAIR-1分子结合。结论:成功地构建了mLAIR-1胞膜外区基因的真核表达载体,表达并纯化了mLAIR-1-Fc融合蛋白。用偶联mLAIR-1-Fc融合蛋白的Sepharose-4B亲和层析柱纯化的兔抗mLAIR-1胞外区抗体,具有高特异性和高效价,为进一步研究mLAIR-1分子的结构和功能提供了新的手段。
AIM： To express and purify the fusion protein of the extraceUular region of murine leukocyte-associated Immunoglobulin-like receptor-1 （mLAIR-1） molecule with Fc fragment of human IgG and to prepare the rabbit antibody against mLAIR-1 extracellular region. METHODS： The eukaryotic expression vector plg/3c-mLAIR-1 was constructed. Rabbits were immunized with the fusion protein purified from the culture medium of mLAIR-1-Fc vector-transfected CHO cells with protein A column. The rabbit antibody against mLAIR-1 extracellular region were purified through Sepharose-4B affinity column coupled with mLAIR-1-Fc. The specificity of the antibody was characterized by indirect fluorescence staining and FCM analysis. RESULTS： The mLAIR-1-Fc fusion protein was expressed and purified. The rabbit antibody against the extracellular region of mLAIR-1 molecule was obtained, which could react with both natural mLAIR-1 molecule on P388D1 cells and recombinant mLAIR- 1 molecule on transfected CHO cells. CONCLUSION： The rabbit antibody against the extracellular region of mLAIR-1is prepared successfully, which may provide a useful tool for studying the structure and function of mLAIR-1.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2005年第5期595-597,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展规划(973)资助(No.2001CB510004)
