Abstract
<title>Abstract</title> <p> Interleukin-6 (IL-6) is a multifunctional cytokine involved in immune regulation and inflammatory responses. However, mammalian cell-based production and functional characterization of recombinant porcine IL-6 remain insufficiently explored. In this study, a recombinant porcine IL-6 expression plasmid was constructed using pcDNA3.1(+) and transiently expressed in Chinese hamster ovary (CHO) cells. Following purification, recombinant porcine IL-6 was obtained at a concentration of 488.25 µg/mL with an endotoxin level of 0.0244 EU/mL. SDS-PAGE, Coomassie Brilliant Blue staining, and Western blot analysis confirmed successful expression and purification of the target protein. Functional evaluation showed that stimulation of porcine alveolar macrophages (3D4/21) with 50 ng/mL recombinant porcine IL-6 for 1 h significantly increased the expression of <italic>SOCS3</italic> , <italic>JUNB</italic> , and <italic>CEBPD</italic> . Transcriptomic analysis identified 61 upregulated and 36 downregulated genes, which were mainly enriched in transcriptional regulation, stress responses, programmed cell death, and immune-related signaling pathways, including IL-17, TNF, MAPK, JAK-STAT, and PI3K-AKT pathways. In addition, recombinant porcine IL-6 promoted scratch closure in B16F10 cells at concentrations of 40 and 80 ng/mL, suggesting potential cross-species cellular responsiveness. Collectively, this study established a CHO-based expression and purification system for recombinant porcine IL-6 and demonstrated its biological activity, providing a valuable resource for cytokine-related immunological and biotechnology studies. </p>