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

Expression of a recombinant lactoferrin N-terminal functional fragment in three expression systems and its efficacy against enterotoxigenic Escherichia coli K88 infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of a recombinant lactoferrin N-terminal functional fragment in three expression systems and its efficacy against enterotoxigenic Escherichia coli K88 infection Wei Li, Aoqian Fu, Wenbin Lu, Jiawei Chen, Baishi Lei, Wuchao Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8094953/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in BMC Veterinary Research → Version 1 posted 12 You are reading this latest preprint version Abstract 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 symptoms 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 symptoms 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. Lactoferrin Pichia pastoris Saccharomyces cerevisiae Escherichia coli Staphylococcus aureus antibacterial activity immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Background Antibiotics have long been used in human health treatments to combat infections. However, the misuse of antibiotics is increasingly leading to problems such as the development of drug resistance and accumulation of drug residues in animal-derived foods[ 1 ]. The development of novel alternatives to antibiotics can help to address these issues. Lactoferrin (LF) is an iron-binding protein of the transferrin family[ 2 ]. Since its first isolation from bovine and human milk in 1960, LF has been the subject of extensive structural and functional studies, demonstrating broad-spectrum activity against bacteria, fungi, and viruses as well as significant antitumor and immunomodulatory effects[ 3 – 5 ]. LF consists of a single polypeptide chain that folds into two largely symmetric and highly homologous globular lobes, referred to as the N-lobe and C-lobe [ 6 ]. The antimicrobial peptides derived from LF are primarily located in the N-terminal region[ 7 ]. These peptides exhibit strong cationic properties, which enable them to damage microbial cell membranes[ 8 ]. Compared with intact proteins, these peptides have a lower molecular weight, which facilitates better absorption by the body and results in stronger antimicrobial effects[ 9 , 10 ]. In 1992, Bellamy et al. isolated a peptide from the N-terminal region of LF, designated as lactoferricin, which demonstrated more than 400 times greater antibacterial activity than intact LF. In addition to its potent antibacterial properties, lactoferricin exhibits multiple biological functions, including antitumor, immunomodulatory, and antioxidant activities[ 11 ]. Colostrum and milk are the richest sources of LF; however, their isolation and purification are costly and yield low quantities, making it difficult to meet the demands of large-scale applications[ 12 ]. The production of LF using recombinant DNA technology in various expression systems is a fundamental approach for addressing these challenges. Commonly used expression systems, such as the Pichia pastoris system (pPICZαA), the Saccharomyces cerevisiae surface display system (pYD1), and the prokaryotic Escherichia coli system (pET-28a), each have their own advantages and limitations. For instance, prokaryotic systems offer high expression yields, but may lack eukaryotic post-translational modifications, which enable glycosylation and facilitate secretory expression or surface display; however, the expression efficiency and protein activity may vary depending on the yeast strain and vector used[ 13 ]. Currently, systematic comparative studies on the expression efficiency and biological activity of LF in different systems are lacking. Enterotoxigenic Escherichia coli (ETEC) K88 is a major pathogen that causes diarrhea in newborn livestock[ 14 ]. Antibiotic treatment is a common treatment approach; however, it readily induces drug resistance and often leads to drug residue accumulation[ 15 ]. LF exhibited activity against traditional antibiotic-resistant strains and was less likely to promote the development of resistance. Although the in vitro antibacterial activity of LF against various bacteria has been reported[ 6 , 16 ], its in vivo protective efficacy against ETEC K88 and its detailed immunoregulatory mechanisms remain incompletely understood. In particular, comprehensive evaluation on the histopathological changes, bacterial loads in organs, and systemic cytokine responses following infection is lacking. In this study, we aimed to compare the in vitro antibacterial activities of the LF expressed using three different expression systems and to evaluate the therapeutic efficacy of the most active recombinant protein in a mouse model infected with ETEC K88. To facilitate secretory expression, we utilized an LF N-terminal functional fragment with a relatively low molecular weight, which included two functional domains (Lfcin: SKCYQWQRRMRKLGAPSITCVRRTS; Lf(1–11): APRKNVRWCAI) as the recombinant expression target[ 10 , 17 ]. The functional domains of the LF N-terminus were expressed using three different expression systems ( P. pastoris, S. cerevisiae, and E. coli) to compare the in vitro antibacterial activities of the expression products. The most active recombinant protein was selected and evaluated in a mouse model infected with ETEC K88 to comprehensively assess its therapeutic efficacy and mechanism of action. This study not only provides a basis for selecting the optimal expression system for the efficient production of highly active LF but also offers robust in vitro and in vivo experimental evidence supporting LF as an effective therapeutic agent against ETEC K88 infection, demonstrating significant theoretical value and application prospects. 2. Materials and methods 2.1. Ethical statement Animal experiments in this study were conducted according to the recommendations of the Chinese Regulations on Laboratory Animals and Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China). The BALB/c mice used in this study were purchased from Beijing Sipeifu Biotechnology Co., Ltd. The experiments were approved by the Animal Welfare and Ethics Committee of the Laboratory Animal Center of Hebei Agriculture University. 2.2 Plasmids, strains, and reagents Vector pPICZαA, vector pET-28a(+), and strain KM71H were preserved by our laboratory. Vector pYD1 and strain EBY100 were purchased from Auno Gene. H5α and Shuffle T7-B competent cells were purchased from Beijing Biomad Biological Technology Co., Ltd. ETEC K88 was obtained from the China Veterinary Microbial Culture Collection Center and S. aureus (ATCC 6538) was purchased from Beijing Beina Chunguang Biotechnology Co., Ltd. The 2×Taq MasterMix was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. (Taizhou);DL2000/DL5000 DNA Marker and SacⅠrestriction enzyme were purchased from TaKaRa Biotechnology (Beijing) Co., Ltd. Protein Marker, polyvinylidene fluoride (PVDF) membrane, and bleomycin were purchased from Beijing Solarbio Science & Technology Co., Ltd. The BIOMIGA Agarose Gel DNA/PCR Product Recovery Kit and BIOMIGA Plasmid Miniprep Kit were purchased from Hangzhou Bio-wisdom Medical Technology Co., Ltd. The Yeast Genomic DNA Rapid Extraction Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. Yeast Extract and tryptone were purchased from OXOID UK7. 2.3 Construction of KM71H-pPICZαA-rLF, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF Based on the LF sequence of Ovis aries (NCBI Accession No. FJ541507.1), a 381-base pair gene fragment encoding the N-terminal functional peptide was selected. After codon optimization for P. pastor is , the gene was synthesized and cloned into the pPICZαA vector, resulting in the construction of the plasmid pPICZαA-rLF. This plasmid was linearized and transformed into P. pastoris KM71H via electroporation. Recombinant yeast strains, designated KM71H-pPICZαA-rLF, were successfully obtained and verified by PCR (using primers AOX1-F/AOX1-R) and DNA sequencing. Using pPICZαA-rLF as the template, the LF fragment was amplified with primers pYD1-rLF-F/R, digested with restriction enzymes, and ligated into the pYD1 vector. The resulting construct was transformed into EBY100 competent cells, and the recombinant strain EBY100-pYD1-rLF was confirmed by colony PCR (primers pYD1-F/R) and sequencing. With pPICZαA-rLF as the template, the LF fragment was amplified using primers 28a-rLF-F/R, digested, and ligated into the pET-28a vector. After transformation into Shuffle T7-B competent cells, the recombinant strain T7-B-pET-28a-rLF was verified using liquid culture PCR (primers T7/T7t). The primers used in this study are listed in Table 1. Table 1 Detect primer Primer name Primer sequence Restriction site AOX1-F 5'-GACTGGTTCCAATTGACAAGC-3' AOX1-R 5'-GGATGTCAGAATGCCATTTGC-3' pYD1-rLF-F 5'-GGTGGTGGTGGTTCT GCTAGC ATGGGTTTGTGTTTG-3' NheI pYD1-rLF-R 5'-GATATCTGCA GAATTC TTAATGATGATGATGATGATGTCTACCCAAAC-3' Eco RⅠ pYD1-F 5'-AGTAACGTTTGTCAGTAATTGC-3' pYD1-R 5'-GTCGATTTTGTTACATCTACA-3' 28a-rLF-F 5'-ATGGGTCGCGGATCC GAATTC ATGGGTTTGTGTTTG-3' Eco RⅠ 28a-rLF-R 5'-CTCGAGTGCGGCCGC AAGCTT TCTACCCAAACCCAT-3' Hin dⅢ T7 5'-TAATACGACTCACTATAGGG-3' T7t 5'-GCTAGTTATTGCTCAGCGG-3' 2.4 Expression and identification of rLF-N-PP, rLF-N-SC, and rLF-N-EC The KM71H-pPICZαA-rLF strain was activated by culturing in YPD liquid medium containing Zeocin, Kan, and Amp at 28 °C for 24 h. Activated cultures were then grown in BMGY medium at 28 °C until the OD600 reached approximately 4. The cells were centrifuged at 2,500 rpm for 5 min, and the pellet was resuspended in BMMY medium and continuously cultured at 28 °C for 4 days, with methanol supplementation every 24 h. The supernatant was collected by centrifugation at 12,000 rpm for 10 min, and 100% TCA was added and mixed thoroughly before storage at –20 °C. The frozen supernatant was centrifuged at 12,000 rpm for 20 min and the protein precipitate was collected. The precipitate was dissolved in a urea-free Binding Buffer, and the samples were subjected to SDS-PAGE and western blot analyses. The expressed protein, designated rLF-N-PP, was dialyzed overnight at 4 °C using pretreated dialysis tubing. Purification was performed using a His-tag protein purification kit according to the manufacturer's instructions and the purified protein was verified by western blotting. The EBY100-pYD1-rLF strain was inoculated into YNB-CAA medium (containing 2% glucose) and cultured overnight at 30 °C with shaking at 250 rpm until the OD₆₀₀ reached 2–5. The cells were harvested by centrifugation at 4,000 rpm for 5 min, the supernatant was discarded, and the pellet was retained. The pellet was resuspended in YNB-CAA medium (containing 2% galactose) and adjusted to an optical density (OD) of 1, followed by induction at 25 °C for 72 h. After induction, cells were collected by centrifugation. The pellet was resuspended in 10 mL pre-chilled PBS and subjected to ice-bath ultrasonication (power 300 W, 3 s ON, 5 s OFF for a total duration of 15 min). The lysate was centrifuged at 4 °C and 12,000 rpm for 30 min. The supernatant (soluble fraction) and pellet (insoluble fraction resuspended in PBS) were collected separately for western blot analysis. The T7-B-pET-28a-rLF strain was cultured in LB/Kan liquid medium at 37 °C until the OD600 reached 0.6–0.8. Expression was induced with 1 mmol/L IPTG for 4 h. The cells were centrifuged at 4 °C, and the pellet was resuspended and sonicated on ice for 15 min. After sonication, the lysate was centrifuged at 4 °C, and both the supernatant and precipitate were sampled for SDS-PAGE analysis. Purification was performed using a His-tagged protein purification kit following the manufacturer's instructions. 2.5 Antibacterial activity of rLF-N-PP, rLF-N-SC, and rLF-N-EC in vitro E. coli and S.aureus glycerol stocks were streaked onto antibiotic-free LB agar plates and incubated at 37 °C for 12 h. A single colony was selected and cultured in antibiotic-free LB liquid medium until the OD600 reached 0.6–0.8. The bacterial culture was mixed with molten LB agar and poured onto plates. After solidification, wells were punched in the agar. Different volumes (50, 100, 150, and 200 μL) of the supernatant were added into the wells, with ampicillin used as a positive control. The plates were then incubated at 37 °C for 12 h. The antibacterial activities of the different proteins were evaluated by measuring the diameters (mm) of the zones of inhibition. 2.6 Thermal stability analysis of rLF-N-PP A 200-μL sample of the culture supernatant was incubated at 95 °C for 0, 10, 20, 40, and 80 min. Residual antibacterial activity against E. coli and S. aureus was determined. 2.7 Determination of antibacterial activity of rLF-N-PP in mice 2.7.1 Animal treatment experiment Fifty 4-week-old female BALB/c mice were randomly divided into five treatment groups: NC (normal saline control), V (empty vector control), F (rLF-N-PP control), K88 (ETEC K88 infection), and K88+F ( rLF-N-PP treatment) ( Table 2). The mice were orally administered the recombinant protein at a dose of 2 mg per mouse for 28 consecutive days. On day 27 of administration, the normal saline control, empty vector control, and rLF-N-PP control groups were intraperitoneally injected with normal saline, whereas the ETEC K88 infection and rLF-N-PP treatment groups were intraperitoneally injected with ETEC K88 (0.5, 2×10 9 CFU/ml). Table 2 Immunization and challenge test groups Group Group Name Number Immunization Protein Challenge A NC 10 Saline Saline B V 10 Empty vector Saline C F 10 rLF-N-PP Saline D K88 10 Saline K88 E K88+F 10 rLF-N-PP K88 2.7.2 Evaluation of histopathological changes Mice that died, became moribund, or lost more than 20% of their body weight were dissected for sample collection. Histopathological evaluations of the intestine, liver, spleen, and kidneys were performed. 2.7.3 E. coli counting assay The number of E. coli cells in the mesentery, liver, spleen, and cecum of the mice was determined using the dilution plate count method. Briefly, tissues were homogenized and serially diluted (10⁴–10⁶ times). A 50-μL aliquot of each diluted suspension was inoculated onto LB agar plates and incubated at 37 °C for 18 h. 2.7.4 Measurement of cytokine (TNF-α, IL-1β, IFN-γ, IL-10) and sIgA concentrations The concentrations of serum cytokines (TNF-α, IL-1β, IFN-γ, IL-10) and secretory immunoglobulin A (sIgA) in the terminal ileum were measured using Jiangsu Enzyme-Linked Immunosorbent Assay (ELISA) kits, following the manufacturer’s instructions. 2.8 Statistical analysis of data The data were statistically analyzed and visualized using GraphPad Prism 8 software. Differences between groups were assessed using t-tests and analyses of variance (ANOVA). The significance levels are denoted as follows: * P < 0.05 (significant), ** P < 0.01 (highly significant), and *** P < 0.001 (extremely significant). 3 Results and analysis 3.1 Construction of KM71H-pPICZαA-rLF, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF Using the genomic DNA of the recombinant strain KM71H-pPICZαA-rLF as a template and primers AOX1-F/AOX1-R, a PCR product of approximately 870 bp was obtained (Fig. 1A). Using the genomic DNA of the recombinant strain EBY100-pYD1-rLF as a template and primers pYD1-F/pYD1-R, a fragment of approximately 729 bp was amplified (Fig. 1B). Using a bacterial culture of the recombinant strain pET-28a-rLF as a template and primers T7/T7t, a PCR product of approximately 686 bp was obtained (Fig. 1C). 3.2 Expression and identification of rLF-N-PP, rLF-N-SC, and rLF-N-EC The KM71H-pPICZαA-rLF positive strain was induced for 96 h, and the supernatant was concentrated and analyzed by western blot. A specific band was observed at approximately 25 kDa (Fig. 2A), consistent with the expected protein size, indicating successful soluble secretory expression of rLF-N-PP. The EBY100-pYD1-rLF strain was subjected to scaled-up cultivation, and the supernatant, pellet, post-sonication supernatant, and post-sonication pellet of both the empty pYD1 vector and LF-N-SC recombinant protein were analyzed by western blotting. The results confirmed the successful expression of rLF-N-SC, which was primarily located in the pellet and post-sonication pellet with a molecular weight of approximately 47.9 kDa, matching the expected size (Fig. 2B). The prokaryotically expressed protein was purified according to the His-tag purification protocol and analyzed by western blot, which revealed a protein size of approximately 19.5 kDa (Fig. 2C). 3.3 Determination of antibacterial activity The results showed that no inhibition zones were observed around recombinant rLF-N-SC (Fig. 3A). In contrast, an inhibition zone surrounded rLF-N-EC (Fig. 3B), with a diameter of 13 mm against ETEC K88 and 11 mm against S. aureus . A clear inhibition zone was evident around rLF-N-PP, with the diameter varying depending on the volume of the supernatant used. No inhibition zones were detected around uninduced or empty vector fermentation supernatants (Fig. 3C). The zone of inhibition assay demonstrated that the eukaryotic protein rLF-N-PP was secreted into the supernatant and exhibited antibacterial activity against all the tested bacterial strains. The recombinant proteins showed stronger inhibitory effects against E. coli than against S.aureus , and the eukaryotic protein (rLF-N-PP) demonstrated superior antibacterial activity compared to the prokaryotic protein (rLF-N-EC). 3.4 Thermal stability analysis Thermal stability tests demonstrated that rLF-N-PP exhibited high thermal stability and maintained antibacterial activity against both E. coli and S.aureus . Stability was not significantly affected by the heating duration (Fig. 4). 3.5 Antibacterial activity of recombinant protein in mice 3.5.1 Clinical symptoms in mice After the challenge, mice in the ETEC K88 group exhibited dull fur, huddling with eyes closed, reduced activity and food intake, decreased sensitivity to external stimuli, and sluggish responses. Mice in the rLF-N-PP treatment group exhibited milder clinical symptoms than those in the ETEC K88 infection group. The control group mice had smooth and glossy fur, normal activity and diet, and displayed no clinical symptoms. To further confirm the infection status, the deceased mice were dissected. Edema and congestion were observed in the intestines and liver. The rLF-N-PP treatment group exhibited milder symptoms than the ETEC K88 infection group, whereas the control group appeared normal with no significant pathological symptoms (Fig. 5). 3.5.2 Gross pathological changes Mice from each group were necropsied, and the organs were collected. Lesions were observed in four major organs: the intestines, liver, spleen, and kidneys. Macroscopically visible pathological changes included intestinal congestion and swelling; enlarged and congested liver with blunt edges and fragile texture prone to rupture; enlarged, rounded, and congested spleen displaying a dark red color; and renal swelling and congestion. The rLF-N-PP treatment group showed less severe lesions than the ETEC K88 infection group, whereas the control group exhibited no significant pathological changes (Fig. 6). 3.5.3 Histopathological changes Further histopathological examination was performed to assess tissue damage. Histopathological studies of HE-stained sections revealed pathological damage in the intestine, liver, spleen, and kidney of the ETEC K88 infection group (Fig. 7); the intestinal mucosal structure was disrupted, with severe villus atrophy and degeneration. Significant shedding of villus and intestinal gland epithelial cells was observed (red arrows). Shed villi and epithelial cells were visible in the intestinal lumen. Numerous villus epithelial cells were separated from the lamina propria (blue arrows), with widened gaps and loosely arranged structures. Muscle cells showed uneven staining, and vacuolar degeneration (blue arrows) and vascular congestion (red arrows) were observed in the liver tissue. Extensive hemorrhage (red arrows) and mild granulocyte infiltration (purple arrows) were observed in the spleen. Occasional renal capsule dilation (light green arrows), hydropic degeneration of renal tubular epithelial cells (green arrows), focal necrosis of renal tubular epithelial cells (black arrows), sloughed epithelial cells in some renal tubular lumens (light blue arrows), sporadic infiltration of granulocytes and lymphocytes (purple arrows), and mild interstitial vascular congestion (red arrows) were observed in the kidney. In contrast, the control group exhibited normal morphology and well-organized structure in all organs, with no significant inflammatory cell infiltration or abnormalities. The rLF-N-PP treatment group showed no significant inflammatory cell infiltration but displayed mild lesions: short and blunted intestinal villi, with minor shedding of villus and gland epithelial cells (red arrows); occasional hepatic vascular congestion (red arrows); mild splenic sinus congestion (red arrows); and slight renal interstitial vascular congestion (red arrows).These histopathological results further demonstrate that rLF-N-PP effectively protected against ETEC K88-induced tissue damage, providing a significant defense against ETEC K88 infection. 3.5.4 Bacterial load in various tissues and organs The number of E. coli cells in the mesenteric lymph nodes of mice in the rLF-N-PP treatment group was significantly lower than that in the ETEC K88 infection group (P < 0.05). The ETEC K88 infection group showed higher bacterial loads in the liver than the rLF-N-PP-treated group. Similarly, the rLF-N-PP treatment group showed a reduced number of E. coli in the spleen compared to the ETEC K88 infection group. The number of E. coli cells in the cecum was higher in the ETEC K88 infection group than in the rLF-N-PP treatment group (Fig. 8). In summary, rLF-N-PP reduced the number of E. coli cells, indicating that it prevented the translocation of E. coli from the abdominal cavity to nearby organs. 3.5.5 Changes in serum TNF-α, IL-1β, IFN-γ, IL-10, and terminal ileum sIgA levels The concentration of the pro-inflammatory cytokine TNF-α in the ETEC K88 infection group was higher than that in the other groups, while rLF-N-PP significantly reduced the TNF-α level (P < 0.05). rLF-N-PP also decreased the concentrations of IL-1β and IFN-γ in the serum. The anti-inflammatory cytokine IL-10 level in the ETEC K88 infection group was lower than that in the other groups, and rLF-N-PP increased IL-10 concentration. Additionally, the terminal ileum sIgA level in the ETEC K88 infection group was higher than that in the other groups, and rLF-N-PP reduced the sIgA concentration (Fig. 9). 4 Discussion ETEC is a significant pathogen causing diarrhea in both humans and young livestock (such as newborn piglets, calves, lambs, and weaned piglets)[ 18 ]. Newborn animals infected with ETEC often suffer from severe watery diarrhea and rapid dehydration, which can lead to death, resulting in high morbidity and mortality rates and substantial economic losses in the livestock industry[ 19 ]. Currently, the treatment of ETEC-related diseases relies primarily on drug therapy. However, in recent years, the misuse of antibiotics has led to serious issues such as drug residues and bacterial resistance, making the search for novel antimicrobial agents that can replace or assist antibiotics particularly urgent[ 20 ]. Therefore, LF, a natural, safe, and resistant antimicrobial protein, has great potential for both research and practical applications. In this study, the plasmids for P. pastoris KM71H-pPICZαA-rLF, S. cerevisiae EBY100-pYD1-rLF, and E. coli T7-B-pET-28a-rLF were successfully constructed and expressed. Bands of the correct size were detected using western blotting. However, significant differences were observed in the in vitro antibacterial activities: rLF-N-PP > rLF-N-EC > rLF-N-SC (inactive). The antibacterial activity of recombinant LF varied significantly across different expression systems, which may be attributed to differences in protein modifications among these systems. The prokaryotic system lacks the post-translational modification capabilities of eukaryotes and cannot glycosylate LF [ 21 ]. Glycosylation is essential to maintain the stability, solubility, and bioactivity of LF, particularly its interactions with bacterial surface receptors[ 22 ]. Furthermore, prokaryotic expression tends to form inclusion bodies, and the refolding process may not fully restore the native conformation, leading to a partial loss of activity[ 23 ]. The complete inactivation of rLF-N-SC may be due to steric hindrance or conformational changes when the active site is displayed on the yeast cell surface, preventing effective binding to bacteria. This suggests that the S. cerevisiae surface display system may be more suitable for vaccine development or screening of binding domains, rather than expressing proteins that require soluble antimicrobial activity. In contrast, the P. pastoris system enables the correct folding and glycosylation of expressed rLF, producing a highly active product that most closely resembles the structure and function of natural LF. Therefore, this study established P. pastoris as the preferred platform for the production of recombinant LF for therapeutic purposes. Li et al. [ 24 ] successfully expressed complete LF from Tibetan sheep in P. pastoris GS115, and the recombinant LF inhibited E. coli and S. aureus growth. Chen et al. [ 25 ] expressed goat LF in P. pastoris X-33 (rGLF), and the iron-binding behavior, papain-inhibiting properties, and thermal stability of the purified rGLF were comparable to those of native goat LF. Although several studies have reported the successful expression of caprine- or ovine-related LF, their investigations were primarily limited to in vitro activity studies. The in vivo results indicated that the therapeutic effect of rLF-N-PP extended far beyond the simple in vitro antibacterial activity, involving a multidimensional, multi-target process. Direct antibacterial effect: The significant reduction in bacterial load across various tissues in treated mice demonstrates that rLF-N-PP effectively inhibits and clears ETEC K88 pathogens in vivo, controlling infection at its source. Immunomodulatory effects: Significant reduction in pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ) and an increase in the anti-inflammatory cytokine (IL-10) indicate that rLF-N-PP effectively modulates the host's immune response to infection, preventing secondary tissue damage caused by a "cytokine storm." This aligns perfectly with the observed reduction in tissue lesions, which is consistent with the findings reported for bovine LF[ 26 ]. Regulation of mucosal immunity: Changes in terminal ileum sIgA levels, the first line of mucosal defense, suggest that rLF-N-PP positively regulates the intestinal mucosal immune barrier, thereby enhancing local defense capabilities. In summary, rLF-N-PP protected mice against ETEC K88 infection by repairing tissue damage, reducing pro-inflammatory cytokine levels, increasing anti-inflammatory cytokine levels, and maintaining intestinal microbiota homeostasis. The antibacterial effect of rLF-N-PP was species-specific, showing greater efficacy against gram-negative bacteria (E. coli) than against gram-positive bacteria ( S. aureus ). One of the primary mechanisms of LF involves its positively charged N-terminal region binding to negatively charged lipopolysaccharides (LPS) on Gram-negative bacteria, disrupting outer membrane integrity ("pore formation") and causing leakage of cellular contents. Gram-positive bacteria lack LPS and are therefore less susceptible to this mechanism[ 27 ]. This characteristic provides a solid theoretical basis for its future application in intestinal infections, primarily caused by gram-negative bacteria (e.g., ETEC and Salmonella). Despite these valuable findings, we acknowledge the limitations of this study. The yield of rLF-N-PP was low and undetectable by SDS-PAGE and structural characterization was insufficient to precisely detail the molecular structures of the three rLF variants, preventing a direct molecular-level explanation of the activity differences. Additionally, the study used a single drug dose and time point, failing to establish a dose-effect relationship or explore the optimal treatment window (e.g., whether preventive administration or post-infection treatment is more effective), leaving room for optimization in future translational applications. All in vivo experiments were based on mouse models, and extrapolation of conclusions to other species requires further validation. Future work will focus on increasing the protein yield and enhancing the therapeutic efficacy of the recombinant protein through strategies such as promoter optimization, signal peptide engineering, molecular chaperone co-expression, fermentation process optimization, and multicopy integration. 5 Conclusions In conclusion, this study demonstrates that rLF-N-PP is a multifunctional protein whose therapeutic effects are achieved through the dual mechanisms of direct antibacterial action and indirect immunomodulation. This provides strong preclinical evidence for the development of LF as a novel antimicrobial/anti-inflammatory agent, with great potential to replace or reduce antibiotic use and address drug-resistant bacterial infections. Abbreviations LF: Lactoferrin ETEC K88: Enterotoxigenic Escherichia coli K88 P. pastoris: Pichia pastoris S. cerevisiae: Saccharomyces cerevisiae E. coli: Escherichia coli TNF-α‌: Tumor necrosis factor-alpha IL-1 β ‌: Interleukin-1 beta IFN- γ ‌: Interferon-gamma IL-10‌: Interleukin-10 sIgA‌: Secretory immunoglobulin A Declarations Ethics approval and consent to participate Animal experiments in this study were conducted according to the recommendations of the Chinese Regulations on Laboratory Animals and Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China). The experiments were approved by the Animal Welfare and Ethics Committee of the Laboratory Animal Center of Hebei Agriculture University. Consent for publication Not applicable. Availability of data and materials The datasets used in this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the Collaborative Research Program between the Shijiazhuang Municipal Government and the Chinese Academy of Agricultural Sciences (CAAS) ( 2524901002A). Authors' contributions WL drafted the manuscript. AQF, WBL and JWC carried out the study, BSL, WCZ, KZ, QYL, XL, JRC and YW conceived the study and participated in its design and coordination. WZY supervised the study, provided critical feedback, and contributed to the manuscript’s final revision. All authors reviewed and approved the final manuscript. Acknowledgements Not applicable. References Tian M, He X, Feng Y, Wang W, Chen H, Gong M, Liu D, Clarke JL, van Eerde A. Pollution by Antibiotics and Antimicrobial Resistance in LiveStock and Poultry Manure in China, and Countermeasures. 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(A) KM71H-pPICZαA-rLF;（B）EBY100-pYD1- rLF;（C）T7-B-pET-28a- rLF. M: DL2000 DNA Marker; Lane 1: PCR product from sample colony.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/0af71a8f3ade6fb0241669c5.png"},{"id":99794775,"identity":"8558ee3b-8d7a-459a-a70c-293d9969ed48","added_by":"auto","created_at":"2026-01-08 13:36:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":192303,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot identification of rLF-N-PP, rLF-N-SC and rLF-N-EC. (A) M: Protein molecular weight Marker; Lane1: rLF-N-PP; Lane 2: Empty vector control; (B) Lane 1-4: Empty vector pYD1(supernatant, precipitation, post-ultrasonic supernatant and post-ultrasonic precipitation), M: Protein molecular weight Marker, Lane 5-8: rLF-N-SC (supernatant, precipitation, post-ultrasonic supernatant, and post-ultrasonic precipitation); (C)M: Protein molecular weight Marker; Lane1: rLF-N-EC; Lane 2: Empty vector control.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/fdfae5474a44bb62b4c0a3a4.png"},{"id":99795315,"identity":"80c9d523-3e6f-49c1-a706-2a652639b5e3","added_by":"auto","created_at":"2026-01-08 13:37:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":495805,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity assay. (A) 1: EBY100 (whole cells); 2: EBY100-pYD1-rLF (whole cells); 3-6: EBY100-pYD1-rLF (supernatant, pellet, sonicated supernatant, sonicated pellet); 7: Ampicillin. (B) 1: Ampicillin; 2: rLF-N-EC. (C) 1: Empty vector supernatant; 2: Ampicillin; 3: 200 μL uninduced supernatant; 4-7: KM71H-pPICZαA-rLF supernatant (50, 100, 150, 200 μL).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/5c1a383ee2cf1a43dccc3d24.png"},{"id":99793074,"identity":"46321deb-0f67-42fb-8762-7da51c2653b7","added_by":"auto","created_at":"2026-01-08 13:30:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":561161,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the thermal stability of rLF-N-PP. (A) Antibacterial activity against E. coli and S. aureus.1: Ampicillin; 2-6: 200 μL supernatant incubated at 95 °C for 0, 10, 20, 40, and 80 min, respectively. (B) Diameter of the inhibition zone of 200 μL culture supernatant. All experiments were performed with at least three replicates.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/5ebaaf2468d150c155e26011.png"},{"id":99794858,"identity":"93e79239-5884-4bda-9fc1-ca48d01dcf3e","added_by":"auto","created_at":"2026-01-08 13:36:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":279814,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic pathological changes in mice across different groups. NC: Normal saline control; V: Empty vector control; F: rLF-N-PP control; K88: ETEC K88 infection; K88+F: rLF-N-PP treatment.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/9244b1c2e1c729911aaac723.png"},{"id":99636712,"identity":"f4b63621-58ae-4c76-87e7-f29ed7630d34","added_by":"auto","created_at":"2026-01-06 17:15:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1032634,"visible":true,"origin":"","legend":"\u003cp\u003ePathological changes in mice across different groups. NC: Normal saline control; V: Empty vector control; F: rLF-N-PP control; K88: ETEC K88 infection; K88+F: rLF-N-PP treatment.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/e7454184ccd1910f579d8942.png"},{"id":99636722,"identity":"f7addf18-c8c6-4b39-8822-216873996869","added_by":"auto","created_at":"2026-01-06 17:15:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1973710,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological changes in mice across different groups. NC: Normal saline control; V: Empty vector control; F: rLF-N-PP control; K88: ETEC K88 infection; K88+F: rLF-N-PP treatment.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/d8ded0ef7a18ec17aab57372.png"},{"id":99794127,"identity":"746335c1-ed70-454d-a0bc-59f0105c8257","added_by":"auto","created_at":"2026-01-08 13:34:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":373624,"visible":true,"origin":"","legend":"\u003cp\u003eThe concentration of E.coli in different tissues and organs. (A) Mesentery; (B)Liver; (C)Spleen; (D)Cecum.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/32402296d06db5abf2f95af4.png"},{"id":99636718,"identity":"6c6ec838-2279-48d2-9b36-898d6a60a49c","added_by":"auto","created_at":"2026-01-06 17:15:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":239500,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of cytokines and terminal ileal sIgA in serum. (A) TNF-α concentration; (B)IL-1β concentration; (C) IFN-γ concentration; (D) IL-10 concentration; (E)Terminal ileum sIgA concentration.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/979b6ed7bf51cfa625a5cf80.png"},{"id":105224477,"identity":"5e3f168c-4c46-403e-9cf2-be7bbf91fcfa","added_by":"auto","created_at":"2026-03-23 16:14:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6396618,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/4b9c170c-4b7d-4664-8b55-26626dee0b46.pdf"},{"id":99795576,"identity":"e4647872-6721-4d8d-acb0-9c4e0a9c8a63","added_by":"auto","created_at":"2026-01-08 13:38:54","extension":"rar","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1685115,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.rar","url":"https://assets-eu.researchsquare.com/files/rs-8094953/v1/8391aed3fcd25a13eff3ce6d.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression of a recombinant lactoferrin N-terminal functional fragment in three expression systems and its efficacy against enterotoxigenic Escherichia coli K88 infection","fulltext":[{"header":"1. Background","content":"\u003cp\u003eAntibiotics have long been used in human health treatments to combat infections. However, the misuse of antibiotics is increasingly leading to problems such as the development of drug resistance and accumulation of drug residues in animal-derived foods[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The development of novel alternatives to antibiotics can help to address these issues. Lactoferrin (LF) is an iron-binding protein of the transferrin family[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Since its first isolation from bovine and human milk in 1960, LF has been the subject of extensive structural and functional studies, demonstrating broad-spectrum activity against bacteria, fungi, and viruses as well as significant antitumor and immunomodulatory effects[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. LF consists of a single polypeptide chain that folds into two largely symmetric and highly homologous globular lobes, referred to as the N-lobe and C-lobe [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The antimicrobial peptides derived from LF are primarily located in the N-terminal region[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These peptides exhibit strong cationic properties, which enable them to damage microbial cell membranes[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Compared with intact proteins, these peptides have a lower molecular weight, which facilitates better absorption by the body and results in stronger antimicrobial effects[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In 1992, Bellamy et al. isolated a peptide from the N-terminal region of LF, designated as lactoferricin, which demonstrated more than 400 times greater antibacterial activity than intact LF. In addition to its potent antibacterial properties, lactoferricin exhibits multiple biological functions, including antitumor, immunomodulatory, and antioxidant activities[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eColostrum and milk are the richest sources of LF; however, their isolation and purification are costly and yield low quantities, making it difficult to meet the demands of large-scale applications[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The production of LF using recombinant DNA technology in various expression systems is a fundamental approach for addressing these challenges. Commonly used expression systems, such as the \u003cem\u003ePichia pastoris\u003c/em\u003e system (pPICZαA), the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e surface display system (pYD1), and the prokaryotic \u003cem\u003eEscherichia coli\u003c/em\u003e system (pET-28a), each have their own advantages and limitations. For instance, prokaryotic systems offer high expression yields, but may lack eukaryotic post-translational modifications, which enable glycosylation and facilitate secretory expression or surface display; however, the expression efficiency and protein activity may vary depending on the yeast strain and vector used[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Currently, systematic comparative studies on the expression efficiency and biological activity of LF in different systems are lacking.\u003c/p\u003e \u003cp\u003eEnterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) K88 is a major pathogen that causes diarrhea in newborn livestock[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Antibiotic treatment is a common treatment approach; however, it readily induces drug resistance and often leads to drug residue accumulation[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. LF exhibited activity against traditional antibiotic-resistant strains and was less likely to promote the development of resistance. Although the in vitro antibacterial activity of LF against various bacteria has been reported[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], its in vivo protective efficacy against ETEC K88 and its detailed immunoregulatory mechanisms remain incompletely understood. In particular, comprehensive evaluation on the histopathological changes, bacterial loads in organs, and systemic cytokine responses following infection is lacking.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to compare the in vitro antibacterial activities of the LF expressed using three different expression systems and to evaluate the therapeutic efficacy of the most active recombinant protein in a mouse model infected with ETEC K88. To facilitate secretory expression, we utilized an LF N-terminal functional fragment with a relatively low molecular weight, which included two functional domains (Lfcin: SKCYQWQRRMRKLGAPSITCVRRTS; Lf(1\u0026ndash;11): APRKNVRWCAI) as the recombinant expression target[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The functional domains of the LF N-terminus were expressed using three different expression systems (\u003cem\u003eP. pastoris, S. cerevisiae, and\u003c/em\u003e E. coli) to compare the in vitro antibacterial activities of the expression products. The most active recombinant protein was selected and evaluated in a mouse model infected with ETEC K88 to comprehensively assess its therapeutic efficacy and mechanism of action. This study not only provides a basis for selecting the optimal expression system for the efficient production of highly active LF but also offers robust in vitro and in vivo experimental evidence supporting LF as an effective therapeutic agent against ETEC K88 infection, demonstrating significant theoretical value and application prospects.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Ethical statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments in this study were conducted according to the recommendations of the Chinese Regulations on Laboratory Animals and Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of the People\u0026rsquo;s Republic of China). The BALB/c mice used in this study were purchased from Beijing Sipeifu Biotechnology Co., Ltd. The experiments were approved by the Animal Welfare and Ethics Committee of\u0026nbsp;the Laboratory Animal Center of Hebei Agriculture University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Plasmids, strains, and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVector pPICZ\u0026alpha;A, vector pET-28a(+), and strain KM71H were preserved by our laboratory. Vector pYD1 and strain EBY100 were purchased from Auno Gene. H5\u0026alpha; and Shuffle T7-B competent cells were purchased from Beijing Biomad Biological Technology Co., Ltd. ETEC K88 was obtained from the China Veterinary Microbial Culture Collection Center and \u003cem\u003eS. aureus\u003c/em\u003e (ATCC 6538) was purchased from Beijing Beina Chunguang Biotechnology Co., Ltd. The 2\u0026times;Taq MasterMix was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. (Taizhou);DL2000/DL5000 DNA Marker and SacⅠrestriction enzyme were purchased from TaKaRa Biotechnology (Beijing) Co., Ltd. Protein Marker, polyvinylidene fluoride (PVDF) membrane, and bleomycin were purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. The BIOMIGA Agarose Gel DNA/PCR Product Recovery Kit and BIOMIGA Plasmid Miniprep Kit were purchased from Hangzhou Bio-wisdom Medical Technology Co., Ltd. The Yeast Genomic DNA Rapid Extraction Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. Yeast Extract and tryptone were purchased from OXOID UK7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Construction of KM71H-pPICZ\u0026alpha;A-rLF, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the LF sequence of \u003cem\u003eOvis aries\u003c/em\u003e (NCBI Accession No. FJ541507.1), a 381-base pair gene fragment encoding the N-terminal functional peptide was selected. After codon optimization for \u003cem\u003eP. pastor\u003c/em\u003e\u003cem\u003eis\u003c/em\u003e, the gene was synthesized and cloned into the pPICZ\u0026alpha;A vector, resulting in the construction of the plasmid pPICZ\u0026alpha;A-rLF. This plasmid was linearized and transformed into \u003cem\u003eP. pastoris\u003c/em\u003e KM71H via electroporation. Recombinant yeast strains, designated KM71H-pPICZ\u0026alpha;A-rLF, were successfully obtained and verified by PCR (using primers AOX1-F/AOX1-R) and DNA sequencing. Using pPICZ\u0026alpha;A-rLF as the template, the LF fragment was amplified with primers pYD1-rLF-F/R, digested with restriction enzymes, and ligated into the pYD1 vector. The resulting construct was transformed into EBY100 competent cells, and the recombinant strain EBY100-pYD1-rLF was confirmed by colony PCR (primers pYD1-F/R) and sequencing. With pPICZ\u0026alpha;A-rLF as the template, the LF fragment was amplified using primers 28a-rLF-F/R, digested, and ligated into the pET-28a vector. After transformation into Shuffle T7-B competent cells, the recombinant strain T7-B-pET-28a-rLF was verified using liquid culture PCR (primers T7/T7t). The primers used\u0026nbsp;in this study are listed in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1 Detect primer\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003ePrimer name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003ePrimer sequence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003eRestriction site\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003eAOX1-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-GACTGGTTCCAATTGACAAGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003eAOX1-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-GGATGTCAGAATGCCATTTGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003epYD1-rLF-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-GGTGGTGGTGGTTCT\u003cu\u003eGCTAGC\u003c/u\u003eATGGGTTTGTGTTTG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003eNheI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003epYD1-rLF-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-GATATCTGCA\u003cu\u003eGAATTC\u003c/u\u003eTTAATGATGATGATGATGATGTCTACCCAAAC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003eEco\u003c/em\u003eRⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003epYD1-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-AGTAACGTTTGTCAGTAATTGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003epYD1-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-GTCGATTTTGTTACATCTACA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003e28a-rLF-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-ATGGGTCGCGGATCC\u003cu\u003eGAATTC\u003c/u\u003eATGGGTTTGTGTTTG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003eEco\u003c/em\u003eRⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003e28a-rLF-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-CTCGAGTGCGGCCGC\u003cu\u003eAAGCTT\u003c/u\u003eTCTACCCAAACCCAT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003eHin\u003c/em\u003edⅢ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003eT7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-TAATACGACTCACTATAGGG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14.6209%;\"\u003e\n \u003cp\u003eT7t\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72.3827%;\"\u003e\n \u003cp\u003e5\u0026apos;-GCTAGTTATTGCTCAGCGG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.9964%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\u003cp\u003e\u003cstrong\u003e2.4 Expression and identification of rLF-N-PP, rLF-N-SC, and rLF-N-EC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe KM71H-pPICZ\u0026alpha;A-rLF strain was activated by culturing in YPD liquid medium containing Zeocin, Kan, and Amp at 28 \u0026deg;C for 24 h. Activated cultures were then grown in BMGY medium at 28 \u0026deg;C until the OD600 reached approximately 4. The cells were centrifuged at 2,500 rpm for 5 min, and the pellet was resuspended in BMMY medium and continuously cultured at 28 \u0026deg;C for 4 days, with methanol supplementation every 24 h. The supernatant was collected by centrifugation at 12,000 rpm for 10 min, and 100% TCA was added and mixed thoroughly before storage at \u0026ndash;20 \u0026deg;C. The frozen supernatant was centrifuged at 12,000 rpm for 20 min and the protein precipitate was collected. The precipitate was dissolved in a urea-free Binding Buffer, and the samples were subjected to SDS-PAGE and western blot analyses. The expressed protein, designated rLF-N-PP, was dialyzed overnight at 4 \u0026deg;C using pretreated dialysis tubing. Purification was performed using a His-tag protein purification kit according to the manufacturer\u0026apos;s instructions and the purified protein was verified by western blotting.\u003c/p\u003e\n\u003cp\u003eThe EBY100-pYD1-rLF strain was inoculated into YNB-CAA medium (containing 2% glucose) and cultured overnight at 30 \u0026deg;C with shaking at 250 rpm until the OD₆₀₀ reached 2\u0026ndash;5. The cells were harvested by centrifugation at 4,000 rpm for 5 min, the supernatant was discarded, and the pellet was retained. The pellet was resuspended in YNB-CAA medium (containing 2% galactose) and adjusted to an optical density (OD) of 1, followed by induction at 25 \u0026deg;C for 72 h. After induction, cells were collected by centrifugation. The pellet was resuspended in 10 mL pre-chilled PBS and subjected to ice-bath ultrasonication (power 300 W, 3 s ON, 5 s OFF for a total duration of 15 min). The lysate was centrifuged at 4 \u0026deg;C and 12,000 rpm for 30 min. The supernatant (soluble fraction) and pellet (insoluble fraction resuspended in PBS) were collected separately for western blot analysis.\u003c/p\u003e\n\u003cp\u003eThe T7-B-pET-28a-rLF strain was cultured in LB/Kan liquid medium at 37 \u0026deg;C until the OD600 reached 0.6\u0026ndash;0.8. Expression was induced with 1 mmol/L IPTG for 4 h. The cells were centrifuged at 4 \u0026deg;C, and the pellet was resuspended and sonicated on ice for 15 min. After sonication, the lysate was centrifuged at 4 \u0026deg;C, and both the supernatant and precipitate were sampled for SDS-PAGE analysis. Purification was performed using a His-tagged protein purification kit following the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Antibacterial activity of rLF-N-PP, rLF-N-SC, and rLF-N-EC in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE. coli and \u003cem\u003eS.aureus\u003c/em\u003e glycerol stocks were streaked onto antibiotic-free LB agar plates and incubated at 37 \u0026deg;C for 12 h. A single colony was selected and cultured in antibiotic-free LB liquid medium until the OD600 reached 0.6\u0026ndash;0.8. The bacterial culture was mixed with molten LB agar and poured onto plates. After solidification,\u0026nbsp;wells were punched in the agar. Different volumes (50, 100, 150, and 200\u0026nbsp;\u0026mu;L) of the supernatant were added into the wells, with ampicillin used as a positive control. The plates were\u0026nbsp;then incubated at 37 \u0026deg;C for 12 h. The antibacterial activities of the different proteins were evaluated by measuring the diameters (mm) of the zones of inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Thermal stability analysis of rLF-N-PP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 200-\u0026mu;L sample of the culture supernatant was incubated at 95 \u0026deg;C for 0, 10, 20, 40, and 80 min. Residual antibacterial activity against E. coli and \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003ewas determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Determination of antibacterial activity of rLF-N-PP in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.1 Animal treatment experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFifty 4-week-old female BALB/c mice were randomly divided into five treatment groups: NC (normal saline control), V (empty vector control), F (rLF-N-PP control), K88 (ETEC K88 infection), and K88+F ( rLF-N-PP treatment) ( Table 2). The mice were orally administered the recombinant protein at a dose of 2 mg per mouse for 28 consecutive days. On day 27 of administration, the normal saline control, empty vector control, and rLF-N-PP control groups were intraperitoneally injected with normal saline, whereas the ETEC K88 infection and rLF-N-PP treatment groups were intraperitoneally injected with ETEC K88 (0.5, 2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/ml).\u003c/p\u003e\n\u003cp\u003eTable 2 Immunization and challenge test groups\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGroup Name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eImmunization Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eChallenge\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSaline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSaline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEmpty vector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSaline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003erLF-N-PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSaline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eK88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSaline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eK88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eK88+F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003erLF-N-PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eK88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.2 Evaluation of histopathological changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice that died, became moribund, or lost more than 20% of their body weight were dissected for sample collection. Histopathological evaluations of the intestine, liver, spleen, and kidneys were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.3 E. coli counting assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number of E. coli cells in the mesentery, liver, spleen, and cecum of the mice was determined using the dilution plate count method. Briefly, tissues were homogenized and serially diluted (10⁴\u0026ndash;10⁶ times). A 50-\u0026mu;L aliquot of each diluted suspension was inoculated onto LB agar plates and incubated at 37 \u0026deg;C for 18 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7.4 Measurement of cytokine (TNF-\u0026alpha;, IL-1\u0026beta;, IFN-\u0026gamma;, IL-10) and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esIgA\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations of serum cytokines (TNF-\u0026alpha;, IL-1\u0026beta;, IFN-\u0026gamma;, IL-10) and secretory immunoglobulin A (sIgA) in the terminal ileum were measured using Jiangsu Enzyme-Linked Immunosorbent Assay (ELISA) kits, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Statistical analysis of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were statistically analyzed and visualized using GraphPad Prism 8 software. Differences between groups were assessed using t-tests and analyses of variance (ANOVA). The significance levels are denoted as follows: * P \u0026lt; 0.05 (significant), ** P \u0026lt; 0.01 (highly significant), and *** P \u0026lt; 0.001 (extremely significant).\u003c/p\u003e\n"},{"header":"3 Results and analysis","content":"\u003cp\u003e\u003cb\u003e3.1 Construction of KM71H-pPICZαA-rLF, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing the genomic DNA of the recombinant strain KM71H-pPICZαA-rLF as a template and primers AOX1-F/AOX1-R, a PCR product of approximately 870 bp was obtained (Fig.\u0026nbsp;1A). Using the genomic DNA of the recombinant strain EBY100-pYD1-rLF as a template and primers pYD1-F/pYD1-R, a fragment of approximately 729 bp was amplified (Fig.\u0026nbsp;1B). Using a bacterial culture of the recombinant strain pET-28a-rLF as a template and primers T7/T7t, a PCR product of approximately 686 bp was obtained (Fig.\u0026nbsp;1C).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Expression and identification of rLF-N-PP, rLF-N-SC, and rLF-N-EC\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe KM71H-pPICZαA-rLF positive strain was induced for 96 h, and the supernatant was concentrated and analyzed by western blot. A specific band was observed at approximately 25 kDa (Fig.\u0026nbsp;2A), consistent with the expected protein size, indicating successful soluble secretory expression of rLF-N-PP. The EBY100-pYD1-rLF strain was subjected to scaled-up cultivation, and the supernatant, pellet, post-sonication supernatant, and post-sonication pellet of both the empty pYD1 vector and LF-N-SC recombinant protein were analyzed by western blotting. The results confirmed the successful expression of rLF-N-SC, which was primarily located in the pellet and post-sonication pellet with a molecular weight of approximately 47.9 kDa, matching the expected size (Fig.\u0026nbsp;2B). The prokaryotically expressed protein was purified according to the His-tag purification protocol and analyzed by western blot, which revealed a protein size of approximately 19.5 kDa (Fig.\u0026nbsp;2C).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Determination of antibacterial activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results showed that no inhibition zones were observed around recombinant rLF-N-SC (Fig.\u0026nbsp;3A). In contrast, an inhibition zone surrounded rLF-N-EC (Fig.\u0026nbsp;3B), with a diameter of 13 mm against ETEC K88 and 11 mm against \u003cem\u003eS. aureus\u003c/em\u003e. A clear inhibition zone was evident around rLF-N-PP, with the diameter varying depending on the volume of the supernatant used. No inhibition zones were detected around uninduced or empty vector fermentation supernatants (Fig.\u0026nbsp;3C). The zone of inhibition assay demonstrated that the eukaryotic protein rLF-N-PP was secreted into the supernatant and exhibited antibacterial activity against all the tested bacterial strains. The recombinant proteins showed stronger inhibitory effects against E. coli than against \u003cem\u003eS.aureus\u003c/em\u003e, and the eukaryotic protein (rLF-N-PP) demonstrated superior antibacterial activity compared to the prokaryotic protein (rLF-N-EC).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.4 Thermal stability analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThermal stability tests demonstrated that rLF-N-PP exhibited high thermal stability and maintained antibacterial activity against both E. coli and \u003cem\u003eS.aureus\u003c/em\u003e. Stability was not significantly affected by the heating duration (Fig.\u0026nbsp;4).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 Antibacterial activity of recombinant protein in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5.1 Clinical symptoms in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter the challenge, mice in the ETEC K88 group exhibited dull fur, huddling with eyes closed, reduced activity and food intake, decreased sensitivity to external stimuli, and sluggish responses. Mice in the rLF-N-PP treatment group exhibited milder clinical symptoms than those in the ETEC K88 infection group. The control group mice had smooth and glossy fur, normal activity and diet, and displayed no clinical symptoms. To further confirm the infection status, the deceased mice were dissected. Edema and congestion were observed in the intestines and liver. The rLF-N-PP treatment group exhibited milder symptoms than the ETEC K88 infection group, whereas the control group appeared normal with no significant pathological symptoms (Fig.\u0026nbsp;5).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5.2 Gross pathological changes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice from each group were necropsied, and the organs were collected. Lesions were observed in four major organs: the intestines, liver, spleen, and kidneys. Macroscopically visible pathological changes included intestinal congestion and swelling; enlarged and congested liver with blunt edges and fragile texture prone to rupture; enlarged, rounded, and congested spleen displaying a dark red color; and renal swelling and congestion. The rLF-N-PP treatment group showed less severe lesions than the ETEC K88 infection group, whereas the control group exhibited no significant pathological changes (Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5.3 Histopathological changes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFurther histopathological examination was performed to assess tissue damage. Histopathological studies of HE-stained sections revealed pathological damage in the intestine, liver, spleen, and kidney of the ETEC K88 infection group (Fig.\u0026nbsp;7); the intestinal mucosal structure was disrupted, with severe villus atrophy and degeneration. Significant shedding of villus and intestinal gland epithelial cells was observed (red arrows). Shed villi and epithelial cells were visible in the intestinal lumen. Numerous villus epithelial cells were separated from the lamina propria (blue arrows), with widened gaps and loosely arranged structures. Muscle cells showed uneven staining, and vacuolar degeneration (blue arrows) and vascular congestion (red arrows) were observed in the liver tissue. Extensive hemorrhage (red arrows) and mild granulocyte infiltration (purple arrows) were observed in the spleen. Occasional renal capsule dilation (light green arrows), hydropic degeneration of renal tubular epithelial cells (green arrows), focal necrosis of renal tubular epithelial cells (black arrows), sloughed epithelial cells in some renal tubular lumens (light blue arrows), sporadic infiltration of granulocytes and lymphocytes (purple arrows), and mild interstitial vascular congestion (red arrows) were observed in the kidney. In contrast, the control group exhibited normal morphology and well-organized structure in all organs, with no significant inflammatory cell infiltration or abnormalities. The rLF-N-PP treatment group showed no significant inflammatory cell infiltration but displayed mild lesions: short and blunted intestinal villi, with minor shedding of villus and gland epithelial cells (red arrows); occasional hepatic vascular congestion (red arrows); mild splenic sinus congestion (red arrows); and slight renal interstitial vascular congestion (red arrows).These histopathological results further demonstrate that rLF-N-PP effectively protected against ETEC K88-induced tissue damage, providing a significant defense against ETEC K88 infection.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5.4 Bacterial load in various tissues and organs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe number of E. coli cells in the mesenteric lymph nodes of mice in the rLF-N-PP treatment group was significantly lower than that in the ETEC K88 infection group (P \u0026lt; 0.05). The ETEC K88 infection group showed higher bacterial loads in the liver than the rLF-N-PP-treated group. Similarly, the rLF-N-PP treatment group showed a reduced number of E. coli in the spleen compared to the ETEC K88 infection group. The number of E. coli cells in the cecum was higher in the ETEC K88 infection group than in the rLF-N-PP treatment group (Fig.\u0026nbsp;8). In summary, rLF-N-PP reduced the number of E. coli cells, indicating that it prevented the translocation of E. coli from the abdominal cavity to nearby organs.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5.5 Changes in serum TNF-α, IL-1β, IFN-γ, IL-10, and terminal ileum sIgA levels\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe concentration of the pro-inflammatory cytokine TNF-α in the ETEC K88 infection group was higher than that in the other groups, while rLF-N-PP significantly reduced the TNF-α level (P \u0026lt; 0.05). rLF-N-PP also decreased the concentrations of IL-1β and IFN-γ in the serum. The anti-inflammatory cytokine IL-10 level in the ETEC K88 infection group was lower than that in the other groups, and rLF-N-PP increased IL-10 concentration. Additionally, the terminal ileum sIgA level in the ETEC K88 infection group was higher than that in the other groups, and rLF-N-PP reduced the sIgA concentration (Fig.\u0026nbsp;9).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eETEC is a significant pathogen causing diarrhea in both humans and young livestock (such as newborn piglets, calves, lambs, and weaned piglets)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Newborn animals infected with ETEC often suffer from severe watery diarrhea and rapid dehydration, which can lead to death, resulting in high morbidity and mortality rates and substantial economic losses in the livestock industry[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Currently, the treatment of ETEC-related diseases relies primarily on drug therapy. However, in recent years, the misuse of antibiotics has led to serious issues such as drug residues and bacterial resistance, making the search for novel antimicrobial agents that can replace or assist antibiotics particularly urgent[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, LF, a natural, safe, and resistant antimicrobial protein, has great potential for both research and practical applications.\u003c/p\u003e\u003cp\u003eIn this study, the plasmids for \u003cem\u003eP. pastoris\u003c/em\u003e KM71H-pPICZαA-rLF, \u003cem\u003eS. cerevisiae\u003c/em\u003e EBY100-pYD1-rLF, and E. coli T7-B-pET-28a-rLF were successfully constructed and expressed. Bands of the correct size were detected using western blotting. However, significant differences were observed in the in vitro antibacterial activities: rLF-N-PP \u0026gt; rLF-N-EC \u0026gt; rLF-N-SC (inactive).\u003c/p\u003e\u003cp\u003eThe antibacterial activity of recombinant LF varied significantly across different expression systems, which may be attributed to differences in protein modifications among these systems. The prokaryotic system lacks the post-translational modification capabilities of eukaryotes and cannot glycosylate LF [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Glycosylation is essential to maintain the stability, solubility, and bioactivity of LF, particularly its interactions with bacterial surface receptors[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, prokaryotic expression tends to form inclusion bodies, and the refolding process may not fully restore the native conformation, leading to a partial loss of activity[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The complete inactivation of rLF-N-SC may be due to steric hindrance or conformational changes when the active site is displayed on the yeast cell surface, preventing effective binding to bacteria. This suggests that the \u003cem\u003eS. cerevisiae\u003c/em\u003e surface display system may be more suitable for vaccine development or screening of binding domains, rather than expressing proteins that require soluble antimicrobial activity. In contrast, the \u003cem\u003eP. pastoris\u003c/em\u003e system enables the correct folding and glycosylation of expressed rLF, producing a highly active product that most closely resembles the structure and function of natural LF. Therefore, this study established \u003cem\u003eP. pastoris\u003c/em\u003e as the preferred platform for the production of recombinant LF for therapeutic purposes.\u003c/p\u003e\u003cp\u003eLi et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] successfully expressed complete LF from Tibetan sheep in \u003cem\u003eP. pastoris\u003c/em\u003e GS115, and the recombinant LF inhibited E. coli and \u003cem\u003eS. aureus\u003c/em\u003e growth. Chen et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] expressed goat LF in \u003cem\u003eP. pastoris\u003c/em\u003e X-33 (rGLF), and the iron-binding behavior, papain-inhibiting properties, and thermal stability of the purified rGLF were comparable to those of native goat LF. Although several studies have reported the successful expression of caprine- or ovine-related LF, their investigations were primarily limited to in vitro activity studies.\u003c/p\u003e\u003cp\u003eThe in vivo results indicated that the therapeutic effect of rLF-N-PP extended far beyond the simple in vitro antibacterial activity, involving a multidimensional, multi-target process. Direct antibacterial effect: The significant reduction in bacterial load across various tissues in treated mice demonstrates that rLF-N-PP effectively inhibits and clears ETEC K88 pathogens in vivo, controlling infection at its source. Immunomodulatory effects: Significant reduction in pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ) and an increase in the anti-inflammatory cytokine (IL-10) indicate that rLF-N-PP effectively modulates the host's immune response to infection, preventing secondary tissue damage caused by a \"cytokine storm.\" This aligns perfectly with the observed reduction in tissue lesions, which is consistent with the findings reported for bovine LF[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Regulation of mucosal immunity: Changes in terminal ileum sIgA levels, the first line of mucosal defense, suggest that rLF-N-PP positively regulates the intestinal mucosal immune barrier, thereby enhancing local defense capabilities. In summary, rLF-N-PP protected mice against ETEC K88 infection by repairing tissue damage, reducing pro-inflammatory cytokine levels, increasing anti-inflammatory cytokine levels, and maintaining intestinal microbiota homeostasis.\u003c/p\u003e\u003cp\u003eThe antibacterial effect of rLF-N-PP was species-specific, showing greater efficacy against gram-negative bacteria (E. coli) than against gram-positive bacteria (\u003cem\u003eS. aureus\u003c/em\u003e). One of the primary mechanisms of LF involves its positively charged N-terminal region binding to negatively charged lipopolysaccharides (LPS) on Gram-negative bacteria, disrupting outer membrane integrity (\"pore formation\") and causing leakage of cellular contents. Gram-positive bacteria lack LPS and are therefore less susceptible to this mechanism[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This characteristic provides a solid theoretical basis for its future application in intestinal infections, primarily caused by gram-negative bacteria (e.g., ETEC and Salmonella).\u003c/p\u003e\u003cp\u003eDespite these valuable findings, we acknowledge the limitations of this study. The yield of rLF-N-PP was low and undetectable by SDS-PAGE and structural characterization was insufficient to precisely detail the molecular structures of the three rLF variants, preventing a direct molecular-level explanation of the activity differences. Additionally, the study used a single drug dose and time point, failing to establish a dose-effect relationship or explore the optimal treatment window (e.g., whether preventive administration or post-infection treatment is more effective), leaving room for optimization in future translational applications. All in vivo experiments were based on mouse models, and extrapolation of conclusions to other species requires further validation. Future work will focus on increasing the protein yield and enhancing the therapeutic efficacy of the recombinant protein through strategies such as promoter optimization, signal peptide engineering, molecular chaperone co-expression, fermentation process optimization, and multicopy integration.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eIn conclusion, this study demonstrates that rLF-N-PP is a multifunctional protein whose therapeutic effects are achieved through the dual mechanisms of direct antibacterial action and indirect immunomodulation. This provides strong preclinical evidence for the development of LF as a novel antimicrobial/anti-inflammatory agent, with great potential to replace or reduce antibiotic use and address drug-resistant bacterial infections.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eLF:\u0026nbsp;\u003c/strong\u003eLactoferrin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETEC K88:\u003c/strong\u003e Enterotoxigenic \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eK88\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP. pastoris:\u0026nbsp;\u003c/strong\u003ePichia pastoris\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS. cerevisiae:\u0026nbsp;\u003c/strong\u003eSaccharomyces cerevisiae\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. coli:\u0026nbsp;\u003c/strong\u003eEscherichia coli\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTNF-\u0026alpha;\u0026zwnj;:\u0026nbsp;\u003c/strong\u003eTumor necrosis factor-alpha\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL-1\u003c/strong\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e\u0026zwnj;:\u0026nbsp;\u003c/strong\u003eInterleukin-1 beta\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFN-\u003c/strong\u003e\u003cstrong\u003e\u0026gamma;\u003c/strong\u003e\u003cstrong\u003e\u0026zwnj;:\u0026nbsp;\u003c/strong\u003eInterferon-gamma\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL-10\u0026zwnj;:\u0026nbsp;\u003c/strong\u003eInterleukin-10\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esIgA\u0026zwnj;:\u0026nbsp;\u003c/strong\u003eSecretory immunoglobulin A\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments in this study were conducted according to the recommendations of the Chinese Regulations on Laboratory Animals and Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of the People\u0026rsquo;s Republic of China). The experiments were approved by the Animal Welfare and Ethics Committee of the Laboratory Animal Center of Hebei Agriculture University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Collaborative Research Program between the Shijiazhuang Municipal Government and the Chinese Academy of Agricultural Sciences (CAAS) ( 2524901002A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026apos; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWL drafted the manuscript. AQF, WBL and JWC carried out the study, BSL, WCZ, KZ, QYL, XL, JRC and YW conceived the study and participated in its design and coordination. WZY supervised the study, provided critical feedback, and contributed to the manuscript\u0026rsquo;s final revision. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTian M, He X, Feng Y, Wang W, Chen H, Gong M, Liu D, Clarke JL, van Eerde A. Pollution by Antibiotics and Antimicrobial Resistance in LiveStock and Poultry Manure in China, and Countermeasures. Antibiot (Basel Switzerland) 2021, 10(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Zhang X, Wu Y, Wang Z, Yu T, Chen P, Tong P, Gao J, Chen H. The Iron Binding Ability Maps the Fate of Food-Derived Transferrins: A Review. J Agric Food Chem. 2024;72(32):17771\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiura K, Nagai Y, Yokouchi A, Miwa K. Expressing recombinant human lactoferrin with antibacterial activity in Nicotiana benthamiana. Plant Biotechnol (Tokyo Japan). 2023;40(1):63\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosa L, Cutone A, Conte MP, Campione E, Bianchi L, Valenti P. An overview on in vitro and in vivo antiviral activity of lactoferrin: its efficacy against SARS-CoV-2 infection. Biometals: Int J role metal ions biology Biochem Med. 2023;36(3):417\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStella MM, Soetedjo R, Tandarto K, Arieselia Z, Regina R. Bovine Lactoferrin and Current Antifungal Therapy Against Candida Albicans: A Systematic Review and Meta-Analysis. 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Front Immunol. 2022;13:885253.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaek KH, Tangchang W, Choi EJ, Lee WK, Lee KH, Lee HK, Byun JW, Son HY. Experimental infection of post-weaned pigs with F18-encoding enterotoxigenic and enterotoxigenic/shigatoxigenic Escherichia coli strain isolated from the diarrheic feces in Korea. Open veterinary J. 2023;13(6):705\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLajunen TJ, Souza Silva L, Sullman MJM. Through the Pharmacist\u0026rsquo;s Lens: A Qualitative Study of Antibiotic Misuse and Antimicrobial Resistance in Brazilian Communities. 2025, 14(11):1074.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang X, Tong Y, Xue W, Yang YF, Chen X, Liu J, Chen D. Expression and characterization of recombinant human lactoferrin in edible alga Chlamydomonas reinhardtii. Biosci Biotechnol Biochem. 2019;83(5):851\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu Y, Zhao S, Liu J, Liu Z, He J, Cao H, Zhao H, Wang C, Jin Y, Qi Y, et al. Assessment of the Conformation Stability and Glycosylation Heterogeneity of Lactoferrin by Native Mass Spectrometry. J Agric Food Chem. 2024;72(17):10089\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu K, Tong Z, Zhang X, Dahmani M, Zhao M, Hu M, Li X, Xue Z. A Review: Development of a Synthetic Lactoferrin Biological System. Biodesign Res. 2024;6:0040.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Zhu W, Luo M, Ren H, Tang L, Liao H, Wang Y. Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system. Protein Exp Purif. 2015;109:35\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen GH, Yin LJ, Chiang IH, Jiang ST. Expression and purification of goat lactoferrin from Pichia pastoris expression system. J Food Sci. 2007;72(2):M67\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakakura N, Wakabayashi H, Yamauchi K, Takase M. Influences of orally administered lactoferrin on IFN-gamma and IL-10 production by intestinal intraepithelial lymphocytes and mesenteric lymph-node cells. Biochem cell biology = Biochimie et Biol cellulaire. 2006;84(3):363\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrago-Serrano ME, de la Garza-Amaya M, Luna JS, Campos-Rodr\u0026iacute;guez R. Lactoferrin-lipopolysaccharide (LPS) binding as key to antibacterial and antiendotoxic effects. Int Immunopharmacol. 2012;12(1):1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lactoferrin, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Staphylococcus aureus, antibacterial activity, immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-8094953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8094953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLactoferrin (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\u0026rsquo;s in vivo protective efficacy against Enterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e 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\u0026mdash;KM71H-pPICZαA-rLF, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF\u0026mdash;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 symptoms 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe 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 \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was lower than that on E. coli.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTreatment with rLF-N-PP improved clinical symptoms 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.\u003c/p\u003e","manuscriptTitle":"Expression of a recombinant lactoferrin N-terminal functional fragment in three expression systems and its efficacy against enterotoxigenic Escherichia coli K88 infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-06 17:15:37","doi":"10.21203/rs.3.rs-8094953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-11T17:38:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-17T15:53:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-16T04:48:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-12T19:29:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87349885198162938820221396083937992392","date":"2026-01-06T02:00:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70779366008849565325209507483731107331","date":"2026-01-05T15:11:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136309534936127861924715934009319107962","date":"2026-01-05T07:59:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-05T06:35:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-21T19:34:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-17T11:18:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-17T03:13:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-11-17T03:10:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"711e7ecd-4092-4fa3-8b78-6b052bdd8ca5","owner":[],"postedDate":"January 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:10:47+00:00","versionOfRecord":{"articleIdentity":"rs-8094953","link":"https://doi.org/10.1186/s12917-026-05398-8","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2026-03-17 15:57:46","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2026-01-06 17:15:37","video":"","vorDoi":"10.1186/s12917-026-05398-8","vorDoiUrl":"https://doi.org/10.1186/s12917-026-05398-8","workflowStages":[]},"version":"v1","identity":"rs-8094953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8094953","identity":"rs-8094953","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}