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

doi:10.12140/j.issn.1000-7423.2025.04.008

Abstract

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

CLC Number:

R383.2

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.

Figures/Tables 5

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References 43

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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