Zhang Jianhua, Liu Shanhong, Shang Zhigang, Shi Li, Yun Jun
Faculty of Biomedical Engineering of Zhengzhou University, Zhengzhou, 450001, Henan Province, People's Republic of China.
Theor Biol Med Model. 2012 Aug 22;9:38. doi: 10.1186/1742-4682-9-38.
We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.
我们通过计算机辅助模拟研究了不同肽接头连接的VH和VL之间的端到端距离与单链抗体活性的关系。首先，我们开发了(G4S)n（其中n = 1 - 9）作为连接VH和VL的接头，并通过同源建模估计单链Fv抗体（scFv）的三维结构。在对分子模型进行评估和优化后，构建每个蛋白质的坐标系并统一到一个坐标系中，然后使用三维空间坐标计算端到端距离。在用(G4S)n（n = 1、3、5、7或9）表达和纯化scFv - n后，通过ELISA测定纯化的ND - 1 scFv - n的免疫反应性。采用多因素关系模型分析影响scFv的结构因素：rn = ABn - ABO2 + CDn - CDO2 + BCn - BCst2。免疫反应性与r值之间的关系表明，当r值 = 3时，融合蛋白结构接近理想状态。随着r值增加，免疫反应性下降，但当r值超过一定阈值时，它趋于稳定。我们使用线性关系分析影响scFv免疫反应性的结构因素。
