Expression of a recombinant lactoferrin N-terminal functional fragment in three expression systems and its efficacy against enterotoxigenic Escherichia coli K88 infection.

BACKGROUND: Lactoferrin (LF) is a multifunctional iron-binding glycoprotein with antimicrobial and immunomodulatory properties. Its significant antimicrobial activity and negligible toxic side effects make it a potential therapeutic agent for antibacterial use. However, LF’s in vivo protective efficacy against Enterotoxigenic Escherichia coli K88 (ETEC K88), a major pathogen that causes diarrhea in newborn livestock, and its detailed immunoregulatory mechanisms remain incompletely understood. To address this knowledge gap, we constructed three engineered strains—KM71H-pPICZαA-rLF, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF—to express the recombinant N-terminal functional fragments of LF, designated as rLF-N-PP, rLF-N-SC, and rLF-N-EC, respectively. They were successfully expressed, identified, and subjected to in vitro antibacterial activity analysis. Furthermore, mice infected with ETEC K88 were treated with rLF-N-PP, and clinical signs and histopathological changes in the intestines, liver, spleen, and kidneys were observed and recorded. Bacterial loads in the mesentery, cecum, liver, and spleen were measured, and levels of serum cytokines (TNF-α, IL-1β, IFN-γ, IL-10) and terminal ileal sIgA were quantified. RESULTS: The results demonstrated successful expression of all three recombinant proteins. In vitro antibacterial assays showed that rLF-N-SC lacked antimicrobial activity, whereas rLF-N-PP exhibited significantly stronger antibacterial activity than rLF-N-EC. The inhibitory effect on Staphylococcus aureus was lower than that on E. coli. CONCLUSIONS: Treatment with rLF-N-PP improved clinical signs in mice infected with ETEC K88; markedly alleviated histopathological damage in the intestines, liver, spleen, and kidneys; reduced bacterial loads in the mesentery, cecum, liver, and spleen; decreased serum levels of pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ) and terminal ileal sIgA; and increased the level of the anti-inflammatory cytokine IL-10 compared to the K88 infection group.