噬菌体抗体库的构建及抗猪脂肪细胞膜单抗的筛选  

Construction of Phage Antibody Library and Screening of Monoclonal Antibodies Specific for Porcine Adipocytes

作　　者：苗向阳[1,2] 丁淑燕[3] 朱玉良[3] 郝慧芳[4] 邵建军[3] 

机构地区：[1]中国农业科学院畜牧研究所 [2]中国农业大学农业生物技术国家重点实验室,北京100094 [3]山东农业大学动物科技学院 [4]中国农业大学农业生物技术国家重点实验室

出　　处：《中国农业科学》2005年第6期1260-1263,共4页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目(30070543);北京市自然科学基金资助项目(5032012)

摘　　要：用猪脂肪细胞以免疫删减法免疫小鼠,取其脾细胞,RT-PCR扩增抗体重链可变区(VH)和轻链可变区(VL)基因,克隆入噬菌粒表达载体pCANTAB5E中,构建单链抗体(ScFv)文库。用猪脂肪细胞,对其进行3轮吸附-洗脱-富集,筛选抗猪脂肪细胞膜单抗。经ELISA检测,51/96克隆具有与猪脂肪细胞结合活性,阳性率为53%,与猪肝细胞则成阴性反应,初步证明了单抗的特异性,为猪脂肪细胞膜单抗的制备提供了一种新的方法。Using subtractive immunization technique, Balb/C mice were immunized with porcine adipocytes, and their spleen cells were harvested. The genes of VH and VL were amplified by RT-PCR and a ScFv phage library of anti-porcine adipocytes was constructed with the amplified V gene. The library was selected using porcine adipocytes as panning antigen by 3 rounds of binding-elution-enrichment procedure. Monoclonal antibodies against porcine adipocytes were acquired by screening. Fifty-one out of 96 randomized clones were demonstrated to have binding capacities to porcine adipocytes, with the positive ratio as much as 53%, while the negative results were observed when reacted with porcine hepatocytes. This strategy may provide an alternative new approach to antibodies.

关 键 词：脂肪细胞膜 噬菌体抗体库 单抗 筛选 构建 猪 ELISA检测 重链可变区 PCR扩增 免疫小鼠 表达载体 单链抗体 结合活性 脾细胞 阳性率 肝细胞 特异性 克隆 轻链 

分 类 号：S852.4[农业科学—基础兽医学] Q782[农业科学—兽医学]