Production and characterization of anti-(mucin MUCI) single-domain antibody in tobacco (Nicotiana tabacum cultivar Xanthi)
Production and characterization of anti-(mucin MUCI) single-domain antibody in tobacco (Nicotiana tabacum cultivar Xanthi)
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DOI：
10.1042/ba20060071
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发表时间：
2007-05-01
影响因子：
2.8
通讯作者：
Memari, Hamid Raiabi
中科院分区：
文献类型：
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作者：
Ismaili, Ahmad;Jalali-Javaran, Mokhtar;Memari, Hamid Raiabi
已知骆驼科成员（骆驼、单峰骆驼、美洲驼、羊驼、原驼和骆马）产生缺乏轻链和CHI（恒定重链结构域）的Ig（免疫球蛋白）。因此，这些重链抗体的抗原特异性结合片段包含一个单一结构域（所谓的“VHH”），并且在生物技术应用中非常重要。为了评估sdAb（单结构域抗体）在植物中的表达和生物活性，由于其小尺寸和抗原识别特性，其将对抗体工程化策略产生重大影响，我们构建了编码对癌症相关粘蛋白MUC 1具有特异性的重组sdAb片段的pBI 121-VHH基因。通过PCR和Western印迹分析转基因烟草（烟草栽培品种Xanthi）植物证明了sdAb的表达，而ELISA结果与各种MUC I抗原和免疫细胞化学与癌细胞系证实了这些分子的活性与亲本重组抗体的活性相比是有利的。通过使用连续的（NH 4）、SO、沉淀、凝胶过滤和免疫亲和层析实现蛋白纯化。通过ELISA对纯化的VHH的分析表明，纯化的抗体片段能够成功地与MUC 1相关肽反应。这些结果再次证实，烟草植物是用于生产具有诊断和治疗应用的正确折叠的VHH抗体片段的合适宿主。
Members of the Camelidae (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce Igs (immunoglobulins) devoid of light chains and CHIs (constant heavy-chain domains). The antigen-specific binding fragments of these heavy-chain antibodies therefore comprise one single domain (the so-called 'VHH') and are of great importance in biotechnological applications. To evaluate the expression and biological activity of sdAbs (single-domain antibodies) in plants, which, on account of their small size and antigen-recognition properties, would have a major impact on antibody-engineering strategies, we constructed a pBI121-VHH gene encoding the recombinant sdAb fragments with specificity for a cancers associated mucin, MUC 1. Analysis of transgenic tobacco (Nicotiana tabacum cultivar Xanthi) plants by PCR and Western blotting demonstrated the expression of sdAb, while ELISA results with various MUC I antigens and immunocytochemistry with, cancerous cell lines confirmed that the activity of these molecules compared favourably with that of the parent recombinant antibodies. Protein purification was achieved by using sequential (NH,),SO, precipitation, gel filtration and immunoaffinity chromatography. Analysis of the purified VHH by ELISA indicated that the purified antibody fragments were able to react successfully with a MUC 1-related peptide. These results reaffirm that the tobacco plant is a suitable host for the production of correctly folded VHH antibody fragments with diagnostic and therapeutic applications.