Valdés Rodolfo, Gómez Leonardo, Padilla Sigifredo, Brito José, Reyes Biunayki, Alvarez Tatiana, Mendoza Otto, Herrera Orlando, Ferro William, Pujol Merardo, Leal Vladimir, Linares Marbelis, Hevia Yasser, García Cristina, Milá Lorely, García Olga, Sánchez Rafael, Acosta Alfonso, Geada Déborah, Paez Rolando, Luis Vega Jorge, Borroto Carlos
Plant Derived Antibody Production and Generation Group, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba.
Biochem Biophys Res Commun. 2003 Aug 15;308(1):94-100. doi: 10.1016/s0006-291x(03)01335-4.
The application of bioengineering to plants for production of biological products for human and animal use has expanded in recent years. The reasons for this expansion are several and include advances in the technology for novel production systems and the need for very large quantities of therapeutic proteins. The process of growing pharmaceutical proteins in plants, extracting, and purifying is a hard task considering the lack of available information concerning these topics. In this work, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen, expressed in stably transformed transgenic Nicotiana tabacum plants, was purified by means of a recombinant protein A Streamline chromatography as the main purification step. The antibody expression level varied with the age of the plants and the number of harvests from 40 to 15microg/ml and the maximum process yield was about 25mg of plantibody/kg of biomass. Protein A Streamline chromatography was successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS-PAGE purity of over 90% but unexpectedly, previous clarification steps could not be totally avoided. The amino acid sequence recognized by this affinity purified plantibody was similar to its murine counterpart verifying the potentiality of plants to replace animals or bioreactors for large-scale production of this monoclonal antibody.
近年来，生物工程在植物中的应用不断拓展，用于生产供人类和动物使用的生物制品。这种拓展有多个原因，包括新型生产系统技术的进步以及对大量治疗性蛋白质的需求。考虑到缺乏关于这些主题的可用信息，在植物中培养药用蛋白质、提取和纯化是一项艰巨的任务。在这项工作中，一种针对乙肝表面抗原的重组鼠单克隆抗体在稳定转化的转基因烟草植物中表达，主要通过重组蛋白A Streamline层析进行纯化。抗体表达水平随植物年龄和收获次数而变化，从40微克/毫升到15微克/毫升不等，最大工艺产量约为25毫克植物抗体/千克生物量。蛋白A Streamline层析成功用于纯化过程，回收率约为60%，植物抗体SDS-PAGE纯度超过90%，但出乎意料的是，之前的澄清步骤无法完全避免。这种亲和纯化的植物抗体识别的氨基酸序列与其鼠源对应物相似，证实了植物在大规模生产这种单克隆抗体方面替代动物或生物反应器的潜力。
