Abstract

Unmethylated cytosine-guanine oligodeoxyribonucleotides (CpG ODNs) are potent immunostimulants via toll-like receptor 9 (TLR9) recognition, but their application is limited by nuclease degradation and high costs. Herein, a designed ssDNA fragment (1826-F) was circularized with T4 DNA ligase and amplified by rolling-circle amplification (RCA) with Phi29 polymerase, self-assembling into RCA-1826 nanoparticles with a mean size of 63.05 nm and a zeta potential of −5.75 mV. Nuclease resistance assays were performed to evaluate degradation stability, showing RCA-1826 degraded slowly over 24 h while linear control 1826-R was fully degraded within 6 h. In vitro , murine macrophages (RAW264.7) and zebrafish liver cells (ZFL) were treated with RCA-1826, and the results demonstrated that inflammatory factor expression was significantly upregulated, verifying its immunostimulatory activity. In vivo , administration of 1 μg RCA-1826 induced robust innate immunity in zebrafish. Combined with inactivated vaccine, RCA-1826 acted as an effective adjuvant, conferring 94.4% protection against Edwardsiella piscicida . This protection rate is 11.1% higher than that of the sulfur-modified ODN1826-adjuvanted vaccine (83.3%). Overall, RCA-based synthesis enables stable, immunogenic CpG DNA nanoparticles, highlighting RCA-1826 as a promising candidate for E. piscicida prevention in aquaculture.