Objective A newly annotated chemokine ligand 8 (CCL8)-like gene was identified and anti-serum prepared to further understand the antiparasitic immunity of Carassius auratus.

Method Based on the previously identified transcripts, the coding sequence of the target gene was cloned via RT-PCR. Bioinformatics analyses were performed to evaluate the physicochemical properties, structure, and phylogenetic relationships. Recombinant protein was expressed using a pET-28a prokaryotic system and purified to immunize Oryctolagus cuniculus for anti-serum preparation followed by titer determination and target protein verification using indirect ELISA and western blot.

Result CCL8-like gene encoded a 93-amino acid polypeptide harboring a typical SCY domain of the chemokine superfamily, with 4 conserved cysteine residues located at positions 33, 34, 57, and 73. The secretory protein shared the highest sequence similarity at 72.53% with that of C. gibelio. The phylogenetic classification of the gene was in the CC chemokine subfamily. A soluble recombinant protein with a molecular weight of approximately 10 kDa was successfully purified that generated rabbit anti-serum recognizing the target protein with a titer exceeding 1∶128 000.

Conclusion The CCL8-like gene in goldfish was first identified and secured with high-purity recombinant protein and anti-serum by this study. It provided the foundation for further studies on the chemokines and antiparasitic immunity in freshwater fish.