关键词: 核酸适配体, 指数富集的配体系统进化技术, CD8, 高通量测序, 人外周血淋巴细胞

Abstract: OBJECTIVE：To identify nucleic acid aptamers that bind specifically to human CD8α molecules. METHODS：A recombinant human CD8α protein with a 6×histidine tag was used as the screening target，and a randomized library of 81 nt was designed for nucleic acid aptamer screening by SELEX technology. The enriched libraries were subjected to high-throughput sequencing and cluster analysis，and the highest abundance sequences were used to verify their specific binding to CD8α protein and CD8⁺ cells by spot hybridization and flow cytometry. The affinity constants (K_ds) of all aptamer candidates were determined using flow cytometry. Finally，normal human peripheral blood lymphocytes were used as a model，and flow cytometry was used to preliminarily evaluate the clinical application of these candidates by prior art to identify CD8⁺ lymphocytes in clinical samples，and to detect whether the binding site of Apt 1 and CD8⁺ lymphocytes was the same as that of CD8 antibody and CD8α protein. RESULTS：The enriched library was sequenced by high-throughput sequencing and 42 803 valid sequence reads were obtained，which were clustered and analyzed by Clustal Omega online software. The top 10 sequences were ranked in order of abundance as the candidate nucleic acid aptamers，which were named as Apt 1-Apt 10，respectively. A highly conserved concordant sequence was present in all candidate sequences (CGTGAGGAGCTTGAAATCC). Among them，Apt 1 had a typical stem-loop structure (ΔG=-12.61 kcal/mol) and bound with high specificity to CD8α protein and CD8⁺ cells. In addition，all 10 sequences bound with high affinity to CD8α protein and CD8⁺ cells，and the affinity constants (K_ds) were at the namor level. Compared with commercial CD8 antibodies and CD8 aptamers disclosed in prior art，the candidate aptamers effectively identified CD8⁺ lymphocytes in human peripheral blood samples，and the binding sites of Apt 1 and CD8⁺ lymphocytes in human peripheral blood samples were different from those of CD8 antibodies. CONCLUSION：Successful screening of nucleic acid aptamers that bound with high affinity and specificity to human CD8α molecules laid the foundation for the subsequent development of clinical diagnostic and therapeutic approaches against CD8 targets.

Key words: nucleic acid aptamer, SELEX, CD8, high-throughput sequencing, human peripheral blood lymphocytes