曼氏血吸虫循环阴极抗原特异性核酸适配体的筛选与鉴定

doi:10.12140/j.issn.1000-7423.2025.04.008

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (4): 497-502.doi: 10.12140/j.issn.1000-7423.2025.04.008

曼氏血吸虫循环阴极抗原特异性核酸适配体的筛选与鉴定

唐琦()(), 仇佳音, 李佳佳, 周新杰, 吕超, 冯婷, 许静, 秦志强^*()()

中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）；传染病溯源预警与智能决策全国重点实验室；国家卫生健康委员会寄生虫病原与媒介生物学重点实验室；世界卫生组织热带病合作中心；科技部国家级热带病国际联合研究中心，上海 200025

收稿日期:2025-04-11 修回日期:2025-05-12 出版日期:2025-08-30 发布日期:2025-10-09
通讯作者: *秦志强（ORCID：0000-0002-1130-468）男，博士，研究员，从事血吸虫感染免疫机理与分子诊断。E-mail：qinzq@nipd.chinacdc.cn
作者简介:唐琦（ORCID：0009-0008-2137-7524），女，硕士研究生，从事寄生虫监测与预警。E-mail：15970005361@163.com
基金资助:
上海市公共卫生体系建设三年行动计划（2023—2025年）重点学科项目(GWVI-11.1-12);国家重点研发计划项目(2021YFC2300800);国家重点研发计划项目(2021YFC2300803)

Selection and identification of aptamers against the circulating cathodic antigen of Schistosoma mansoni

TANG Qi()(), QIU Jiayin, LI Jiajia, ZHOU Xinjie, LV Chao, FENG Ting, XU Jing, QIN Zhiqiang^*()()

National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research; National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; NHC Key Laboratory on Parasite and Vector Biology; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China

Received:2025-04-11 Revised:2025-05-12 Online:2025-08-30 Published:2025-10-09
Contact: *E-mail: qinzq@nipd.chinacdc.cn
Supported by:
Key Discipline Projects under the Shanghai Three-Year Action Plan for Strengthening Public Health System Construction (2023-2025)(GWVI-11.1-12);National Key Research and Development Program of China(2021YFC2300800);National Key Research and Development Program of China(2021YFC2300803)

摘要/Abstract

摘要：

目的筛选曼氏血吸虫循环阴极抗原（circulating cathodic antigen，CCA）特异性核酸适配体，并进行鉴定。方法构建曼氏血吸虫循环阴极抗原的重组原核表达载体（pET-28a-CCA），诱导pET-28a-CCA蛋白表达并通过Ni-NTA柱纯化，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析pET-28a-CCA蛋白的表达情况。以pET-28a-CCA蛋白为靶标，进行指数富集配基的系统进化（SELEX）筛选，利用荧光定量PCR监测分析每一轮文库富集情况。经过多轮加压筛选，获得的产物通过高通量测序分析、生物信息学分析，并采用微量热泳动技术表征（MST）测定候选核酸适配体与pET-28a-CCA蛋白的结合亲和力，进行分子对接。结果双酶切鉴定和测序结果显示，pET-28a-CCA重组质粒插入约为1 044 bp的CCA基因片段。SDS-PAGE电泳结果显示，pET-28a-CCA蛋白的相对分子质量（M_r）约为43 600；SELEX筛选结果显示，第13轮筛选文库达到最佳富集。根据测序结果筛选出的9条候选核酸适配体序列，经过MST检测，有7条候选核酸适配体与pET-28a-CCA蛋白有结合亲和力。TQ-apt1的吉布斯自由能最低（ΔG = -13.09 kcal/mol），GC含量最高（59.20%）、熔解温度最高（102.3 ℃）以及G-四链体得分最高（15分），TQ-apt1与pET-28a-CCA蛋白的亲和力最高，解离常数为2.1 nmol/L。结论成功获得了pET-28a-CCA蛋白的核酸适配体。

关键词: 曼氏血吸虫, 循环阴极抗原, 核酸适配体, 指数富集配基的系统进化技术, 重组原核蛋白

Abstract:

Objective To screen and identify the aptamer specifically targeting the circulating cathodic antigen (CCA) of Schistosoma mansoni. Methods The recombinant prokaryotic expression vector pET-28a-CCA was constructed, and the expression of pET-28a-CCA protein was induced and purified by Ni-NTA column. The expression characteristics of pET-28a-CCA were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using pET-28a-CCA protein as the target, systematic evolution of ligands by exponential enrichment (SELEX) was performed. Quantitative real-time PCR was used to monitor and analyze each round of library enrichment. After multiple rounds of stringent selection, the obtained products were subjected to high-throughput sequencing analysis, bioinformatics analysis, and the characterization of binding affinity of candidate aptamers to the pET-28a-CCA protein using microscale thermophoresis (MST), followed by molecular docking. Results Double enzyme digestion and sequencing results confirmed that the pET-28a-CCA recombinant plasmid contained an approximately 1 044 bp CCA gene fragment. SDS-PAGE results demonstrated successful expression of the pET-28a-CCA protein, with a relative molecular mass (M_r) of approximately 43 600. The SELEX screening results showed that the library of the 13th screening round reached the best enrichment. Based on sequencing results, nine candidate aptamer sequences were selected. Among them, seven aptamers exhibited binding affinity to the pET-28a-CCA as determined by MST analysis. TQ-apt1 had the lowest Gibbs free energy (ΔG = -13.09 kcal/mol), the highest GC content (59.2%), the highest melting temperature (102.3 ℃), and the highest G-quadruplex score (15 points). TQ-apt1 had the highest affinity with pET-28a-CCA protein with a dissociation constant of 2.1 nmol/L. Conclusion In this study, aptamers targeting the pET-28a-CCA protein were successfully obtained through SELEX technology.

Key words: Schistosoma mansoni, Circulating cathodic antigen, Aptamers, Systematic evolution of ligands by exponential enrichment, Recombinant prokaryotic protein

中图分类号:

R383.2

唐琦, 仇佳音, 李佳佳, 周新杰, 吕超, 冯婷, 许静, 秦志强. 曼氏血吸虫循环阴极抗原特异性核酸适配体的筛选与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(4): 497-502.

TANG Qi, QIU Jiayin, LI Jiajia, ZHOU Xinjie, LV Chao, FENG Ting, XU Jing, QIN Zhiqiang. Selection and identification of aptamers against the circulating cathodic antigen of Schistosoma mansoni[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(4): 497-502.

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名称Name	随机区域序列（5'→3'） Random region sequence (5'→3')	GC含量/% Content of GC/%	熔解温度/℃ Melting temperature/℃	G-quadruplex score G-四链体得分
TQ-apt1	TTCAGCACTCCACGCATAGCCGGACGAGGTGGAAACATCCGAGCTGGCGTC-TGAGCCCTATGCGTGCTACCGTGAA	59.20	102.3	15
TQ-apt2	TTCAGCACTCCACGCATAGCAACTGGCATCGGTAGATATGAGTGCTTGACC-TGTCGCCTATGCGTGCTACCGTGAA	52.60	98.4	0
TQ-apt3	TTCAGCACTCCACGCATAGCCAATACGCGGTAGGATGAGTGTGCTGATTAT-TTTGGCCTATGCGTGCTACCGTGAA	50.00	97.3	0
TQ-apt4	TTCAGCACTCCACGCATAGCCCGGTAGGAGTCCAGATTATGTGACTAGAGT-GCGCACCTATGCGTGCTACCGTGAA	53.90	97.7	0
TQ-apt5	TTCAGCACTCCACGCATAGCAAACGCGAGCGGTAGTTAGGAGTGCCTTGCT-GTTTGCCTATGCGTGCTACCGTGAA	53.90	99.1	0
TQ-apt6	TTCAGCACTCCACGCATAGCCCGACACCACGGTGAGGCTGATACAAATTTG-TCGTGCCTATGCGTGCTACCGTGAA	53.90	100.1	0
TQ-apt7	TTCAGCACTCCACGCATAGCCACAAGATACCGGTAGTGTTGAGTGCGTACC-TTTGGCCTATGCGTGCTACCGTGAA	52.60	97.7	0
TQ-apt8	TTCAGCACTCCACGCATAGCAGGCCTCATCCGGTAGTCCTAGAGTGCGATA-TGGAACCTATGCGTGCTACCGTGAA	53.90	97.8	0
TQ-apt9	TTCAGCACTCCACGCATAGCACGATCAAAATGTGCTGCCGTCCTCGGTTGA-GTCTGCCTATGCGTGCTACCGTGAA	53.90	100.5	0

曼氏血吸虫循环阴极抗原特异性核酸适配体的筛选与鉴定

Selection and identification of aptamers against the circulating cathodic antigen of Schistosoma mansoni

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