Heidari Hamid Reza, Bandehpour Mojgan, Vahidi Hossein, Barar Jaleh, Kazemi Bahram, Naderi-Manesh Hossein
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. ; Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2015 Winter;14(1):189-201.
Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand (TRAIL) as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV (Cauliflower Mosaic Virus) helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay (Methylthiazol Tetrazolium Assay). The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable.
分子农业已被视为生产生物制药的一种安全且经济的方法。人肿瘤坏死因子相关凋亡诱导配体（TRAIL）作为一种有前景的生物制药候选物已在不同的表达宿主中产生。然而，尽管植物表达系统有诸多优势，但TRAIL的分子农业却很少受到关注。因此，在本研究中，利用根癌农杆菌LBA 4404在烟草中解决了TRAIL的细胞质生产问题。首先，使用PCR技术在构建的人cDNA文库上获得所需的编码序列。随后，通过亚克隆到35S-CaMV（花椰菜花叶病毒）辅助载体和最终的0179-pGreen表达载体中，提供了TRAIL在植物细胞系统中表达的必要条件。然后，将最终的TRAIL-pGreen表达载体克隆到根癌农杆菌LBA 4404中。随后，通过共培养方法转化烟草细胞，并通过蛋白质免疫印迹分析确认TRAIL的表达。最后，通过色谱技术提取重组TRAIL，并通过MTT法（噻唑蓝比色法）评估其生物活性。蛋白质免疫印迹分析结果表明，只能从烟草细胞中提取到单体和氧化二聚体形式的TRAIL。此外，提取的TRAIL缺乏三聚体组装，降低了其在敏感A549细胞系中的生物活性。总之，尽管烟草细胞能够成功产生TRAIL，但TRAIL的正确组装和功能却不理想。
