Rafighdoust Hediyeh, Ahangarzadeh Shahrzad, Yarian Fatemeh, Taheri Ramezan Ali, Lari Arezou, Bandehpour Mojgan, Salahshoor Dahr Mona
Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
Iran J Basic Med Sci. 2020 Aug;23(8):1053-1058. doi: 10.22038/ijbms.2020.44007.10318.
We previously conducted an research on the interactions between the ribosome display-selected single chain variable fragment (scFv) and factor H binding protein (fHbp) of . We found that heavy chain variable (VH) fragment of this scFv had considerable affinity to fHbp. These results led us to evaluate the ability of this small antibody fragment in binding and detection of fHbp antigen.
In this study, at first, the three-dimensional structure of VH fragment was simulated by Kotai Antibody Builder web server. By using ClusPro 2.0 web server, the 3D structure of the soluble form of fHbp (PDB: 2KC0) was docked to the modeled VH fragment to extract the structure of the complex's binding. Molecular dynamics (MD) simulation was carried out using GROMACS 4.5.3 package for 65 ns. Secondly, coding sequence of VH fragment was cloned separately and expressed in . After purification of the VH fragment, its binding activity to fHbp protein was analyzed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) method.
Important amino acids involved in antigen- antibody interaction were identified by analyzing the fHbp-VH complex. The ability of the VH antibody fragment to bind and detect fHbp antigen has been confirmed by the results of analysis, ELISA and SPR methods.
These results showed that this small fragment of antibody could be used for designing diagnostic kits.
我们之前进行了一项关于核糖体展示筛选的单链可变片段（scFv）与[具体病原体]的因子H结合蛋白（fHbp）之间相互作用的研究。我们发现该scFv的重链可变区（VH）片段与fHbp具有相当的亲和力。这些结果促使我们评估这个小抗体片段结合和检测fHbp抗原的能力。
在本研究中，首先，通过Kotai Antibody Builder网络服务器模拟VH片段的三维结构。利用ClusPro 2.0网络服务器，将fHbp可溶性形式的三维结构（PDB：2KC0）与建模的VH片段对接，以提取复合物结合的结构。使用GROMACS 4.5.3软件包进行65纳秒的分子动力学（MD）模拟。其次，分别克隆VH片段的编码序列并在[具体宿主]中表达。纯化VH片段后，通过酶联免疫吸附测定（ELISA）和表面等离子体共振（SPR）方法分析其与fHbp蛋白的结合活性。
通过分析fHbp-VH复合物，确定了参与抗原-抗体相互作用的重要氨基酸。VH抗体片段结合和检测fHbp抗原的能力已通过[具体分析方法]、ELISA和SPR方法的结果得到证实。
这些结果表明，这个小抗体片段可用于设计诊断试剂盒。
