Ismaili Ahmad, Jalali-Javaran Mokhtar, Rasaee Mohammad J, Rahbarizadeh Fatemeh, Forouzandeh-Moghadam Mehdi, Memari Hamid Rajabi
Department of Plant Breeding, School of Agriculture, Tarbiat Modarres University, Jalale-Ale-Ahmad High Way, Tehran 14115-336, Iran.
Biotechnol Appl Biochem. 2007 May;47(Pt 1):11-9. doi: 10.1042/BA20060071.
Members of the Camelidae (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce Igs (immunoglobulins) devoid of light chains and CH1s (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 cancer-associated mucin, MUC1. Analysis of transgenic tobacco (Nicotiana tabacum cultivar Xanthi) plants by PCR and Western blotting demonstrated the expression of sdAb, while ELISA results with various MUC1 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 (NH4)2SO4 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 MUC1-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.
骆驼科动物（骆驼、单峰驼、美洲驼、羊驼、原驼和小羊驼）的成员已知会产生不含轻链和CH1（恒定重链结构域）的免疫球蛋白（Ig）。因此，这些重链抗体的抗原特异性结合片段仅包含一个结构域（即所谓的“VHH”），在生物技术应用中具有重要意义。为了评估单结构域抗体（sdAb）在植物中的表达和生物学活性，由于其体积小和抗原识别特性，这将对抗体工程策略产生重大影响，我们构建了一个pBI121-VHH基因，该基因编码对癌症相关粘蛋白MUC1具有特异性的重组sdAb片段。通过PCR和蛋白质印迹法对转基因烟草（烟草品种Xanthi）植株进行分析，证实了sdAb的表达，而使用各种MUC1抗原的ELISA结果以及对癌细胞系的免疫细胞化学分析证实，这些分子的活性与亲本重组抗体相比具有优势。通过依次使用硫酸铵沉淀、凝胶过滤和免疫亲和层析实现了蛋白质纯化。通过ELISA对纯化的VHH进行分析表明，纯化的抗体片段能够与MUC1相关肽成功反应。这些结果再次证实，烟草植株是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。
