Abstract：
Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE .12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes.
摘要：
白细胞介素(IL)-23抑制剂单克隆抗体在治疗自身免疫性疾病中显示出显著的功效。DNA或RNA适体表现出与抗体相当的特异性，具有成本效益，非免疫原性，并且没有批次到批次的变化。本研究旨在表征靶向人IL-23的单链DNA(ssDNA)适体。克隆IL-23的α亚基(P19)和完整的IL-23,表达,最后通过Ni2-亚氨基二乙酸亲和色谱纯化蛋白质。通过指数富集(SELEX)使用配体的蛋白质系统进化进行针对P19的ssDNA适体的选择和表征。进行点印迹测定以监测SELEX轮的适体输出的结合。P19蛋白。斑点印迹试验结果阳性的适体的解离常数(Kd),使用ELISA方法基于它们与IL-23的结合来确定。重组P19和IL-23蛋白分别为26和72kDa，分别,在SDS-PAGE上观察到.12%。通过斑点印迹测定监测来自7、8、9、10、11和12轮SELEX的适体输出。揭示来自第8轮的适体具有更强的发光信号并且被选择用于TA克隆。在分析来自克隆的生物素化适体后，对斑点印迹和ELISA中的阳性克隆进行测序。最后,Kd计算显示三个适体具有高亲和力，命名为A23P3、A23P6和A23P15，Kd值为1.37、2.139和2.88nM，分别。本研究结果介绍了三种特异性抗IL-23ssDNA适体，具有高亲和力，可用于治疗和诊断目的。
