School of Life Science, Jilin University, Changchun, Jilin 130012, China.
National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, Jilin 130012, China.
Biomed Res Int. 2017;2017:8946935. doi: 10.1155/2017/8946935. Epub 2017 Sep 28.
The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.
氧化型低密度脂蛋白受体-1（LOX-1）靶向单链可变片段（scFv）是一种有前途的分子，可用于靶向递送至动脉粥样硬化疾病的成像和治疗分子；然而，其应用受到固有低抗原亲和力的限制。在这项研究中，构建了抗 LOX-1 scFv 的三维（3D）模型，并对其与 LOX-1 蛋白的对接进行了研究。为了提高 LOX-1 结合活性，将抗 LOX-1 scFv 设计为在其 N 端和 C 端以及连接区与三个 LOX-1 结合七肽中的一个融合，这三个 LOX-1 结合七肽与抗 LOX-1 scFv 具有不同的 LOX-1 结合界面，通过一系列计算方法进行了分析。这些 scFv/肽融合物被构建，在宿主中成功表达，并通过两步柱纯化过程进行纯化。研究了这些融合蛋白的抗原结合活性、结构特征、热稳定性和血清稳定性。结果表明，N 端融合肽蛋白的 scFv 表现出增强的 LOX-1 结合活性，而稳定性没有降低。这些发现将有助于提高 LOX-1 靶向 scFv 在基于 LOX-1 的治疗中的应用效果。
