Molecular Adjuvant Potential of GCSF and MCSF in Starry Flounder Challenged with Streptococcus parauberis

In fish, the innate immune system is crucial for rapid defense against pathogens. In this study, we performed transcriptome sequencing using next-generation sequencing (NGS) to identify and characterize granulocyte colony-stimulating factor (GCSF) and macrophage colony-stimulating factor (MCSF) in starry flounder (Platichthys stellatus). The GCSF gene (594 bp, 198 aa) features a conserved IL-6 domain, while the MCSF gene (621 bp, 207 aa) contains a predicted transmembrane region. Phylogenetic analysis confirmed high evolutionary conservation with other marine species. Quantitative real-time PCR revealed that GCSF is highly expressed in the skin, peripheral blood leukocytes, and muscle, with significant up-regulation in immune organs following Streptococcus parauberis infection; MCSF exhibited a similar tissue-specific expression pattern. Recombinant GCSF (rGCSF) was produced using a cell-free system and effectively enhanced leukocyte phagocytic activity at an optimal concentration of 150 μg/mL, without causing cytotoxicity in hemolytic assays. In contrast, rMCSF exhibited folding issues during purification. These findings highlight the potential of rGCSF as a molecular adjuvant to enhance immune responses in aquaculture. This study provides foundational knowledge for developing cytokine-based adjuvants, which could reduce antibiotic dependency and enhance vaccine efficacy in sustainable aquaculture systems.