HCMV PP65抗原核酸适配子的筛选及活性鉴定

Screening and activity identification of ssDNA aptamer targeting human cytomegalovirus PP65

目的以人巨细胞病毒HCMV PP65抗原为靶分子,筛选与其有高亲和力、高特异性的适配子。方法采用SELEX技术筛选HCMV PP65抗原特异的适配子,进行亲和性检测以及二级结构预测,以筛选获得的最优序列作为捕获适配子,建立基于"磁珠-适配子-抗原-荧光单克隆抗体"的夹心检测方法,实现对PP65抗原的初步检测。结果本实验经过8轮筛选获得6条与靶物质高亲和性,高特异性结合的适配子。结论适配子的成功筛选,为建立基于适配子检测HCMV PP65抗原的新方法奠定了基础。

Objective To screen ssDNA oligonucleotide aptamers targeting human cytomegalovirus （HCMV） PP65 antigen with high affinity and specificity. Methods The systematic evolution of ligands by exponential enrichment （SELEX） method was used for screening specific aptamers targeting HCMV PP65 antigen. The affinity of the aptamers was detected by DNAMAN software. Then, the sequence with the ELISA and their secondary structures were predicted by the highest affinity to PP65 was used as the capture aptamer to establish a sandwich assay based on magnetic bead-aptamer-antigen-fluorescence labeled monoclonal antibodies for detecting PP65 antigen. Results A total of 6 aptamers with high affinity and specificity to PP65 molecule were obtained by 8 cycles of SELEX. Conclusion PP65 antigen detection method has been established A good foundation for developing aptamer-based HCMV