| 米尔比霉素肟化物ScFv噬菌体展示抗体库的构建 |
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| 引用本文： | 张晓,张睛晴,温爽,刘媛,刘贤进.米尔比霉素肟化物ScFv噬菌体展示抗体库的构建[J].免疫学杂志,2008,24(6):688-691. |
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| 作者姓名： | 张晓 张睛晴 温爽 刘媛 刘贤进 |
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| 作者单位： | 江苏省农业科学院食品质量安全检测研究所; |
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| 基金项目： | 国家自然科学基金项目(30471155);;农业部“948”项目(2005-Z24)资助 |
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| 摘 要： | 目的构建具16元大环内酯共性结构小分子物质的特异性抗体库。方法免疫原MILO-BSA免疫小鼠后从脾细胞中提取总RNA,RT-PCR扩增得到全套抗体重链可变区基因(VH)及轻链可变区基因(VL),SOE-PCR将VH、VL片段拼接扩增得到单链抗体可变区基因片段(ScFv)。将ScFv基因克隆到噬菌粒载体pCANTAB5E中,电转化感受态大肠杆菌,辅助噬菌体超感染得到上清液即为噬菌体抗体库。结果RT-PCR扩增出长360bp左右的抗体重链可变区基因(VH)及340bp左右的轻链可变区基因(VL),SOE-PCR扩增得到750bp左右的ScFv基因片段,成功构建了库容量为2.4×106,滴度为2.0×1012pfu/mL的噬菌体单链抗体库。结论构建的抗体库目的片段连接率较高,多样性较好,为下一步16元大环内酯类小分子物质高特异性抗体的筛选奠了基础。
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| 关 键 词： | 米尔比霉素肟化物 单链可变区抗体 噬菌体展示抗体库 |
Construction of ScFv phage display antibody library against milbemycin oxime |
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| ZHANG Xiao,ZHANG Qing-qing,WEN Shuang,LIU Yuan,LIU Xian-jin.Construction of ScFv phage display antibody library against milbemycin oxime[J].Immunological Journal,2008,24(6):688-691. |
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| Authors: | ZHANG Xiao ZHANG Qing-qing WEN Shuang LIU Yuan LIU Xian-jin |
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| Institution: | Institute of Food Quality and Food Safety;Jiangsu Academy of Agricultural Sciences;Nanjing 210014;China |
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| Abstract: | Objective To construct a specific phage display library for panning antibodies against compounds of 16-membered macrocyclic backbone. Methods The total RNA was extracted from spleen cells of mouse immunized with milbemycin oxime (MILO)-BSA. RT-PCR was used to amplify immunoglobulin variable heavy chain region genes (VH) and variable light chain region genes (VL). The purified VH and VL genes were jointed together through splicing by overlap extension (SOE),using the fragment-containing parts of the linker. ... |
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| Keywords: | Milbemycin oxime Single-chain variable fragment Phage display antibody library |
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