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Published on: October 12, 2018
Rolling circle amplification-based self-assembled CpG nanoparticles: Immune activation and prevention of Edwardsiella
Wenmei Li1, Chenchen Guo1, Kaixin Wu1
1State Key Laboratory of Bioreactor Engineering, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, East China University of Science and Technology, Shanghai, 200237, China.
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.
