Gene cloning of S100β and NGF and localization of their expression in the small intestine of broilers of various ages.

S100 calcium-binding protein β (S100β) and nerve growth factor (NGF) are jointly involved in the function and homeostasis of the enteric nervous system (ENS) in enteric glial cells (EGCs); however, there is limited information on the biological analysis and distributional expression of S100β and NGF in the broiler intestine in relation to age. This study aimed to investigate the biological and expression characteristics of S100β and NGF in the small intestine of broilers at various ages. The duodenum, jejunum, and ileum of 7, 20, 40, 55, and 70 d yellow-feathered broilers were sampled. The full sequences of CDS region of NGF and S100β were cloned for homology comparison, and the biological properties of the proteins were analyzed using different bioinformatics methods. The expression of NGF andS100β was detected by immunohistochemistry, WB and RT-qPCR. The CDS regions of the NGF and S100β genes encode 239 and 92 amino acid residues, respectively, which encode basic unstable hydrophilic transmembrane and acidic stable hydrophilic non-transmembrane proteins, respectively. Both proteins have phosphorylation sites. Homology analysis showed that NGF and S100β was first clustered with Gallus gallus. Immunohistochemical (IHC) staining showed that S100β protein expression in the small intestine peaks at 55 days of age, and NGF protein expression reaches its highest level at 40 days of age. Both proteins are primarily localized in the mucosal epithelium, intestinal glands, and submucosal plexus across the three segments of the small intestine. Western blot (WB) results corroborated the IHC findings, with both proteins exhibiting an initial increase followed by a decline in expression levels. In conclusion, the CDS region of NGF and S100β genes in the small intestine of yellow-feathered broiler chickens was successfully cloned, which was highly conserved during the evolutionary process, and the expression of NGF and S100β proteins in the small intestine showed a pattern of increasing and then decreasing with the age, and they were mainly distributed in the mucous epithelium, intestinal glands, and submucosal plexus of the small intestine. It is suggested that NGF and S100β may be involved in the differentiation and developmental process of EGCs, and EGCs may exert intestinal regulatory functions by secreting NGF and S100β and binding to the corresponding receptors. Provide a theoretical basis for the development and regulatory function of EGCs in the gut.