Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum
Cloning and Expression of TNF Related Apoptosis Inducing Ligand in Nicotiana tabacum
复制标题
DOI：
10.22037/ijpr.2015.1610
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
H. Heidari;M. Bandehpour;H. Vahidi;J. Barar;B. Kazemi;H. Naderi-manesh
中科院分区：
文献类型：
--
作者：
H. Heidari;M. Bandehpour;H. Vahidi;J. Barar;B. Kazemi;H. Naderi-manesh
分子农业已被认为是一种安全和经济的生物制药生产方法。人肿瘤坏死因子相关凋亡诱导配体（TRAIL）作为一种有前途的生物药物候选物，已在不同的表达宿主中产生。然而，尽管植物表达系统具有许多优点，但很少关注TRAIL的分子农业。因此，在本研究中，使用根癌农杆菌LBA 4404在烟草中处理TRAIL的细胞质生产。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35 S-CaMV（花椰菜花叶病毒）辅助载体和最终的0179-pGreen表达载体中，提供了在植物细胞系统中表达TRAIL的必要条件。将最终的TRAIL-pGreen表达载体克隆入A.根瘤菌LBA 4404。随后，N.通过共培养方法转化烟草细胞，并通过蛋白质印迹分析证实TRAIL的表达。最后，通过层析技术提取重组TRAIL，并通过MTT法（Methylthiazol Tetrazolium Assay）检测其生物学活性。Western blot分析结果表明，从N.烟草细胞此外，缺乏提取的TRAIL的三聚体组装降低了其在敏感的A549细胞系中的生物活性。最后，虽然N.烟草细胞可以成功地产生TRAIL，适当的组装和功能的TRAIL是不利的。
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.