Alizadeha Ali Akbar, Hamzeh-Mivehroud Maryam, Haddad Elnaz, Haddad Nazanin, Sharifi Mehdi, Mohammadi Samin, Pourtaghi-Anvarian Samira, Dastmalchi Siavoush
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
A. A. A. and M. H. M. contributed equally to this work.
Iran J Pharm Res. 2019 Spring;18(2):759-771. doi: 10.22037/ijpr.2019.1100646.
Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. The current study aimed to investigate the binding abilities of I44 and I49 domain antibodies to TNF-α. The dAbs were expressed in bacterial expression system and purified by affinity chromatography using Ni-sepharose column. The expression and purity of the proteins were evaluated using western blotting and SDS-PAGE techniques, respectively. ELISA experiment showed that I44 and I49 dAbs bind to TNF-α with the binding constants (K) of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively. The inhibitory effect of dAbs on TNF-α biological effect was determined in MTT assay in which I44 and I49 prevented TNF-α cell cytotoxicity with IC values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-α dAbs could bind to and inhibit TNF-α activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-α. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-α antibodies.
肿瘤坏死因子α（TNF-α）是一种炎症细胞因子，在炎症性疾病的发病机制中起关键作用。本研究旨在研究I44和I49结构域抗体与TNF-α的结合能力。这些单域抗体（dAbs）在细菌表达系统中表达，并使用镍琼脂糖柱通过亲和层析进行纯化。分别使用蛋白质印迹法和SDS-PAGE技术评估蛋白质的表达和纯度。酶联免疫吸附测定（ELISA）实验表明，I44和I49单域抗体与TNF-α结合，结合常数（K）分别为5.18±1.41和2.42±0.55μM。在MTT试验中测定了单域抗体对TNF-α生物学效应的抑制作用，其中I44和I49分别以6.61和3.64μM的半数抑制浓度（IC）阻止了TNF-α的细胞毒性。鉴定出的抗TNF-α单域抗体可以结合并抑制TNF-α的活性。单域抗体的活性可归因于它们与TNF-α建立氢键以及疏水接触的能力。本研究结果可为进一步的结构研究铺平道路，以便引入新的、更有效的抗TNF-α抗体。
