Zhao Zhi-Jun, Chen Jing-Tao, Yuan Jia-Ying, Yin Xiao-Xiang, Song Hua-Yong, Wang Xin-Chun
Department of Oncology, The First Hospital Affiliated to Henan University Kaifeng, China.
Department of Ultrasound Diagnosis, The Directly Under The Hospital of Henan Military Region Zhengzhou, China.
Int J Clin Exp Med. 2015 Sep 15;8(9):14827-36. eCollection 2015.
The purposes was to establish optimal modeling of single-chain antibody molecules based on similarity algorithm and seek the connecting peptides that had the minimal effect on the structure and bioactivity of the variable region of heavy chain (VH) and that of light chain (VL) in a single-chain antibody against liver cancer. After the Linker with different lengths (n=0~7) had been added into single chain fragment variable (ScFv), modeling of the overall sequences of VH, VL and ScFv were conducted respectively. Meanwhile, the peptide chain structure of (Gly4Ser)n was adopted for the connecting peptide. Then the spatial spherical shell layer alignment algorithm based on spherical polar coordinates was utilized for comparing the structural similarity of VH and VL before and after adding connecting peptide. Equally, in order to determine the stability of VH and VL, MATLAB was applied for analysis of the fore and aft distances and the diffusion radius. Indirect ELISA method was used to detect single-chain antibody immunological activity of Linker with different lengths. The MTT assay was utilized for the examination of the inhibition rate of single-chain antibody with different lengths of Linker to liver cancer cell. When n=4, the structural similarity between VH together with VL and their original ones was the highest. When n=3, the influence of connecting peptide on the stability of VH and VL was minimum. When n>3, the fore and aft distances changed little due to the increase and fold of the length of peptide chain. The results of ELISA detection showed that when n=4, affinity of single chain antibody to liver cancer cells was much higher. The MTT test also indicated that when n=4, the inhibition rate of the connecting peptide on hepatoma carcinoma cell reached the highest, and that came second when n=3. When n=4, the structural stability and biological functions of anti-hepatoma single-chain antibody were both favorable. This study has provided a basis for the design and construction of single-chain antibody.
目的是基于相似性算法建立单链抗体分子的最佳模型，并寻找对肝癌单链抗体中重链可变区（VH）和轻链可变区（VL）的结构和生物活性影响最小的连接肽。将不同长度（n = 0~7）的接头添加到单链可变片段（ScFv）中后，分别对VH、VL和ScFv的整体序列进行建模。同时，连接肽采用（Gly4Ser）n的肽链结构。然后利用基于球极坐标的空间球壳层比对算法比较添加连接肽前后VH和VL的结构相似性。同样，为了确定VH和VL的稳定性，应用MATLAB分析前后距离和扩散半径。采用间接ELISA法检测不同长度接头的单链抗体免疫活性。利用MTT法检测不同长度接头的单链抗体对肝癌细胞的抑制率。当n = 4时，VH与VL及其原始结构之间的结构相似性最高。当n = 3时，连接肽对VH和VL稳定性的影响最小。当n>3时，由于肽链长度的增加和折叠，前后距离变化不大。ELISA检测结果表明，当n = 4时，单链抗体对肝癌细胞的亲和力更高。MTT试验也表明，当n = 4时，连接肽对肝癌细胞的抑制率最高，n = 3时次之。当n = 4时，抗肝癌单链抗体的结构稳定性和生物学功能均良好。本研究为单链抗体的设计与构建提供了依据。
