Lactoferrin and anti-inflammatory peptide fusion protein as well as related biological material and application thereof

Abstract

The invention relates to the technical field of bioengineering, and particularly discloses a lactoferrin and anti-inflammatory peptide fusion protein as well as a related biological material and application thereof. The lactoferrin and anti-inflammatory peptide fusion protein provided by the invention has an amino acid sequence as shown in SEQ ID NO: 1. The fusion protein not only completely retains the bacteriostatic activity of lactoferrin, but also integrates the anti-inflammatory function of the anti-inflammatory peptide. The fusion protein not only shows a good antibacterial effect in in-vivo and in-vitro experiments, but also further shows excellent immunomodulatory effect and anti-inflammatory activity in in-vivo experiments. The fusion protein can be used as a novel green and efficient biological agent to replace or partially replace antibiotics, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a lactoferrin-anti-inflammatory peptide fusion protein and its related biomaterials and applications. Background Technology

[0002] Lactoferrin is a multifunctional iron-binding glycoprotein widely found in the secretions of mammals, including milk, saliva, and tears. It possesses various biological functions, including antibacterial, antiviral, anti-inflammatory, and immunomodulatory effects. Anti-inflammatory peptides are a class of small molecule polypeptides with anti-inflammatory activity that can regulate the body's inflammatory response and inhibit bacterial growth. In recent years, with the increasing severity of antibiotic resistance due to overuse, lactoferrin and anti-inflammatory peptides, as green and highly effective antibacterial substances, have shown broad application prospects in replacing antibiotics.

[0003] Although the individual functions of lactoferrin and anti-inflammatory peptides have been extensively studied, current technologies have significant limitations in their fusion expression. First, there are numerous types of lactoferrin and anti-inflammatory peptides (e.g., lactoferrin has bovine and human variants, while anti-inflammatory peptides include various types such as LL-37 and Defensin), and different types exhibit significant differences in their antibacterial spectrum and activity. This makes it difficult to directly extend the optimization of single components to fusion systems. Second, there are few systematic studies on the tandem expression of lactoferrin and anti-inflammatory peptides. Most literature focuses on individual expression or simple combination, lacking genetic engineering-level fusion construction. It is impossible to know whether the fusion protein retains or enhances the original antibacterial and anti-inflammatory functions, or produces unpredictable antagonistic effects. Because existing methods rarely provide effective expression systems for related fusion proteins, it is impossible to assess whether the fusion of specific types of lactoferrin and anti-inflammatory peptides will produce synergistic effects. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a lactoferrin-anti-inflammatory peptide fusion protein and its related biomaterials and applications. This fusion protein exhibits excellent immunomodulatory and anti-inflammatory activity.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions:

[0006] In a first aspect, the present invention provides a fusion protein of lactoferrin and anti-inflammatory peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] The fusion protein provided by this invention is formed by linking ovis lactoferrin (LF) and anti-inflammatory peptide (AIP) using G4Slinker flexible peptide as the linking sequence. This fusion protein has the dual biological activities of lactoferrin and anti-inflammatory peptide, and has high antibacterial activity and immunomodulatory effect.

[0008] Secondly, the present invention provides a recombinant expression vector that can express the lactoferrin and anti-inflammatory peptide fusion protein provided in the first aspect.

[0009] Preferably, the recombinant expression vector uses a eukaryotic expression vector as its backbone.

[0010] Thirdly, the present invention provides a recombinant strain comprising the recombinant expression vector provided in the second aspect.

[0011] Preferably, the recombinant strain uses yeast as the starting strain.

[0012] For example, the yeast includes Pichia pastoris or Saccharomyces cerevisiae, preferably Pichia pastoris.

[0013] Fourthly, the present invention provides the application of the lactoferrin and anti-inflammatory peptide fusion protein provided in the first aspect in the preparation of antibacterial drugs.

[0014] Furthermore, the antibacterial drug includes at least one of the following: a drug that inhibits Gram-negative bacteria, a drug that inhibits Gram-positive bacteria, or a drug that simultaneously inhibits Gram-negative and Gram-positive bacteria.

[0015] Preferably, the Gram-negative bacteria include Escherichia coli.

[0016] Preferably, the Gram-positive bacteria include Staphylococcus aureus.

[0017] More preferably, the lactoferrin and anti-inflammatory peptide fusion protein provided by the present invention can be used to prepare drugs that inhibit Gram-negative bacteria and treat bacterial infectious diseases caused by Gram-negative bacteria, including Escherichia coli.

[0018] Fifthly, the present invention provides the application of the recombinant expression vector provided in the second aspect in the preparation of antibacterial drugs.

[0019] In a sixth aspect, the present invention provides the application of the recombinant strain provided in the third aspect in the preparation of antibacterial drugs.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. This invention is the first to synthesize a fusion protein rLF-AIP by tandem expression of lactoferrin rLF and the anti-inflammatory peptide AIP. This fusion protein not only fully retains the antibacterial activity of lactoferrin but also integrates the anti-inflammatory function of the anti-inflammatory peptide. This fusion protein not only exhibits good antibacterial effects in in vitro and in vivo experiments but also demonstrates excellent immunomodulatory and anti-inflammatory activities in in vivo experiments.

[0022] 2. This invention utilizes bioengineering technology to construct three eukaryotic expression vectors, pPICZαA-rLF, pPICZαA-rLF-AIP, and pYD1-rLF, and one prokaryotic expression vector, pET-28a-rLF. Based on these vectors, four engineered bacterial strains—KM71H-pPICZαA-rLF, KM71H-pPICZαA-rLF-AIP, EBY100-pYD1-rLF, and T7-B-pET-28a-rLF—were constructed, and four corresponding recombinant proteins were obtained. In in vitro experiments, except for the recombinant protein pYD1-rLF, which showed no antibacterial activity, the other three recombinant proteins exhibited antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*. The antibacterial activity of the two eukaryotic recombinant proteins, pPICZαA-rLF and pPICZαA-rLF-AIP, was superior to that of the prokaryotic recombinant protein, with the recombinant protein pPICZαA-rLF-AIP showing the best antibacterial activity.

[0023] 3. In vivo experiments demonstrated that recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP have protective effects against ETEC K88-infected mice, with pPICZαA-rLF-AIP showing superior protective efficacy. pPICZαA-rLF-AIP not only significantly reduced bacterial load in the mesentery and liver, and decreased bacterial load in the cecum and spleen, but also significantly reduced serum levels of pro-inflammatory factors TNF-α and IL-1β, and significantly increased levels of the anti-inflammatory factor IL-10. This indicates that the fusion protein pPICZαA-rLF-AIP of the present invention possesses not only antibacterial activity but also immunomodulatory and anti-inflammatory capabilities.

[0024] 4. The lactoferrin and anti-inflammatory peptide fusion protein of the present invention is a novel, green and efficient biological agent that can be used as an antibiotic alternative and has important application value. Attached Figure Description

[0025] Figure 1 This is a gel electrophoresis image of the recombinant strain KM71H-pPICZαA-rLFPCR identification in Example 1 of this invention;

[0026] Figure 2 This is a gel electrophoresis image of the recombinant strain KM71H-pPICZαA-rLF-AIP PCR identification in Example 1 of this invention;

[0027] Figure 3 This is the SDS-PAGE detection result of the recombinant strain KM71H-pPICZαA-rLF-AIP induced in Example 1 of this invention;

[0028] Figure 4 This is a comparison chart of Western blot detection results for recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP in Example 1 of this invention. Figure 4 A indicates that after induction by the recombinant strain KM71H-pPICZαA-rLF, Figure 4 B indicates that the recombinant strain KM71H-pPICZαA-rLF-AIP was induced;

[0029] Figure 5 These are gel electrophoresis images of the pET-28a empty vector strain and the pET-28a-rLF recombinant strain before induction, after induction, after lysis supernatant, and after precipitation in Comparative Example 1 of this invention. In these images, M represents the protein molecular weight standard, lane 1 represents pET-28a empty vector before induction, lane 2 represents pET-28a empty vector after induction, lane 3 represents pET-28a-rLF before induction, lane 4 represents pET-28a-rLF after induction, lane 5 represents pET-28a-rLF after lysis supernatant, and lane 6 represents pET-28a-rLF after lysis precipitation.

[0030] Figure 6 This is an SDS-PAGE assay result of recombinant strains EBY100-pYD1 and EBY100-pYD1-rLF after induction, culture, and purification in Comparative Example 2 of this invention; where M represents the protein molecular weight standard, lane 1 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1, lane 2 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1, lane 3 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1 after sonication, lane 4 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1 after sonication, lane 5 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1-rLF, lane 6 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF, lane 7 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication, and lane 8 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication;

[0031] Figure 7This is a comparison of the Western blot results of recombinant strains EBY100-pYD1 and EBY100-pYD1-rLF after induction, culture, and purification in Comparative Example 2 of this invention. In the figure, M represents the protein molecular weight standard, lane 1 represents the pYD1 empty vector supernatant, lane 2 represents the pYD1 empty vector precipitate, lane 3 represents the pYD1 empty vector supernatant after sonication, lane 4 represents the pYD1 empty vector precipitate after sonication, lane 5 represents the pYD1-rLF recombinant protein supernatant, lane 6 represents the pYD1-rLF recombinant protein precipitate, lane 7 represents the pYD1-rLF recombinant protein supernatant after sonication, and lane 8 represents the pYD1-rLF recombinant protein precipitate after sonication.

[0032] Figure 8 The results of the antibacterial activity assays of recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP against Escherichia coli and Staphylococcus aureus in Example 1 of this invention are as follows: Figure 8 A represents the recombinant protein pPICZαA-rLF. Figure 8 B represents the recombinant protein pPICZαA-rLF-AIP;

[0033] Figure 9 The results of the assay of the antibacterial activity of pYD1-rLF expressed by Saccharomyces cerevisiae EBY100 against Escherichia coli and Staphylococcus aureus in Example 1 of the present invention are as follows:

[0034] Figure 10 This is the result of the assay of the antibacterial activity of recombinant protein pET28a-rLF against Escherichia coli and Staphylococcus aureus in Example 1 of the present invention. Figure 10 A represents the recombinant protein pET28a-rLF. Figure 10 B is the negative control;

[0035] Figure 11 This is a diagram showing the overall pathological changes in infected mice in Example 2 of the present invention;

[0036] Figure 12 These are pathological changes in organs such as the intestine, liver, spleen, and kidney in mice from different groups in Example 2 of the present invention.

[0037] Figure 13 These are HE staining images of the intestinal, liver, spleen, and kidney tissues of mice in different groups in Example 2 of the present invention.

[0038] Figure 14 This refers to the statistical results of bacterial load in the mesentery, liver, spleen, and cecum of mice in different groups in Example 2 of the present invention; among them, Figure 14 A represents the statistical results of Escherichia coli load in the mesentery of the K88 group, F+K88 group, and FA+K88 group. Figure 14B represents the statistical results of Escherichia coli load in the livers of the K88 group, F+K88 group, and FA+K88 group. Figure 14 C represents the statistical results of Escherichia coli load in the spleen of the K88 group, F+K88 group, and FA+K88 group. Figure 14 D represents the statistical results of Escherichia coli load in the cecum of the K88 group, F+K88 group, and FA+K88 group;

[0039] Figure 15 These are the statistical results of cytokines in the serum of mice from different groups in Example 2 of the present invention; wherein, Figure 15 A represents the statistical results of serum levels of the pro-inflammatory factor TNF-α in mice from different groups. Figure 15 B represents the statistical results of serum levels of the pro-inflammatory factor IL-1β in mice from different groups. Figure 15 C represents the statistical results of IFN-γ levels in the serum of mice from different groups. Figure 15 D represents the statistical results of the anti-inflammatory factor IL-10 in the serum of mice in different groups. Figure 15 E represents the statistical results of sIgA, a mucosal defense molecule, in the serum of mice from different groups. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] 1. The main reagents used in this invention and their preparation methods are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] The primers used in this invention are shown in Table 2.

[0046] Table 2

[0047]

[0048] Note: The underlined area is the restriction enzyme cleavage site.

[0049] 2. The Western Blot verification method for recombinant proteins in this invention is as follows:

[0050] (1) Preparation of protein samples

[0051] Take 30 μL of the purified protein from the recombinant bacteria, add 10 μL of 4x protein loading buffer, boil in water for 10 min, and centrifuge at 12000 rpm for 2 min at 4℃.

[0052] (2) SDS-PAGE electrophoresis

[0053] After cleaning and leak testing the glass plates, fix them on the gel casting apparatus. Quickly add the separating gel and stacking gel between the glass plates according to Table 10. Insert the comb and remove it after 10 minutes. Install the prepared gel into the vertical electrophoresis tank, add 1xSDS buffer solution, add 10μL of sample to each well, and perform SDS-PAEG electrophoresis.

[0054] (3) Protein transfer

[0055] Wet transfer method: Soak the sponge pad and filter paper in the wet transfer solution beforehand. Cut the PVDF membrane according to the size of the gel. Before use, soak the PVDF membrane in anhydrous ethanol for 2 minutes to activate the membrane. If there are air bubbles when assembling the sandwich layer, remove them with a scraper. The membrane transfer should be carried out on ice at 200mA for 2 hours.

[0056] (4) Closed

[0057] Place the PVDF membrane in 5 mL of 5% skim milk powder solution and incubate at room temperature for 1 hour;

[0058] (5) Incubation of primary antibody

[0059] All antibodies were diluted with 5% skim milk solution. His protein was incubated with His-tag primary antibody at a dilution of 1:2500 at room temperature for 1 hour at a speed of 30-50 rpm. After incubation, the antibody was washed four times with an appropriate amount of TBST for 5 minutes each time. Secondary antibody was incubated at a dilution of 1:5000 at room temperature for 1 hour at a speed of 30-50 rpm. The antibody was then washed four times with an appropriate amount of TBST for 5 minutes each time.

[0060] (6) Exposure

[0061] Add an appropriate amount of ECL luminescent liquid to the membrane, expose it under a gel imaging system, and analyze the results.

[0062] Example 1

[0063] This embodiment provides a lactoferrin and anti-inflammatory peptide fusion protein having the amino acid sequence shown in SEQ ID NO:1. The specific construction method is as follows:

[0064] 1. Construction of recombinant eukaryotic expression vectors

[0065] 1.1 Primer Design and Synthesis

[0066] Based on the principles of enzyme digestion, ligation, and homologous recombination in PCR and molecular construction, primers for amplifying target fragments on different vectors were designed using Primer 5.0 software. The primer sequences are shown in Table 2. Among them, F, R1, R2, and R3 were used to amplify the target fragment of pPICZαA-rLF-AIP; in addition, two pairs of universal primers targeting the pPICZαA and pYD1 plasmids were synthesized respectively.

[0067] 1.2 Construction of recombinant expression plasmid pPICZαA-rLF

[0068] The amino acid sequence of rLF is shown in SEQ ID NO:2. The nucleotide sequence corresponding to this amino acid sequence was optimized to use codons preferred by Pichia pastoris. The optimized rLF coding gene is shown in SEQ ID NO:3. Sangon Biotech (Shanghai) Co., Ltd. (hereinafter referred to as Sangon) was commissioned to synthesize the above coding gene into the pPICZαA vector. After PCR identification and sequencing alignment, the recombinant expression plasmid pPICZαA-rLF was obtained.

[0069] The gel electrophoresis results obtained by PCR showed that the target gene was 870 bp in size, which is consistent with the coding gene of rLF.

[0070] 1.3 Construction of recombinant strain KM71H-pPICZαA-rLF

[0071] (1) Plasmid linearization

[0072] The recombinant expression plasmid pPICZαA-rLF was linearized, and the enzyme digestion system is shown in Table 3 below.

[0073] Table 3

[0074]

[0075]

[0076] (2) Transformation into KM71H host bacteria

[0077] The KM71H strain was streaked onto YPD solid medium and incubated overnight. Single colonies were then picked and cultured until OD500. 600After being washed twice with an appropriate amount of ddH2O, 100 μL of 1 mol / L sorbitol was added; the linearized recombinant plasmid was added to KM71H competent cells, electroporated (1500V, 4.8ms), 1 mL of 1 mol / L sorbitol was added, and the mixture was incubated on ice for 5 min and then cultured at 28℃ and 200 rpm for 1 h; the supernatant was discarded by centrifugation and plated on YPD solid medium to obtain the recombinant strain; the genome of the recombinant strain was extracted using a bio-engineered yeast genome extraction kit, and PCR amplification and identification were performed. The strain containing the positive genome was the recombinant strain KM71H-pPICZαA-rLF.

[0078] The genome of the recombinant strain KM71H-pPICZαA-rLF was used as a template, and AOX1-F and AOX1-R were used as primers for PCR identification. The PCR reaction system is shown in Table 4.

[0079] Table 4

[0080] Reagent Name volume 2×Taq Mix 10.0μL AOX1-F 1.0μL AOX1-R 1.0μL pPICZαA-rLF genome 2.0μL <![CDATA[Sterilized ddH2O]]> 6.0μL Total volume 20.0μL

[0081] PCR reaction conditions: 94℃ pre-denaturation for 5 min; followed by 32 cycles, each cycle consisting of: 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s; and a final extension at 72℃ for 10 min.

[0082] Gel electrophoresis image of PCR identification is shown below Figure 1 As shown. By Figure 1 It can be seen that the target gene is about 870bp in size, which is consistent with the coding gene of rLF.

[0083] 1.4 Construction of recombinant expression plasmid pPICZαA-rLF-AIP

[0084] (1) Obtaining the target gene fragment rLF-AIP

[0085] The gene encoding the anti-inflammatory peptide (AIP) with the amino acid sequence shown in SEQ ID NO:4 (e.g., SEQ ID NO:5) was fused into the recombinant plasmid pPICZαA-rLF. The specific steps are as follows: Using the recombinant plasmid pPICZαA-rLF as the backbone, a G4Slinker flexible peptide was added between the anti-inflammatory peptide and lactoferrin sequences to ensure that their tertiary structures do not interfere with each other, thus preserving the biological activity of each protein. Using the recombinant plasmid pPICZαA-rLF as a template and primers F and R1, R2 and R3, the target fragment rLF-AIP of the recombinant expression plasmid pPICZαA-rLF-AIP was obtained by PCR. The PCR amplification system is shown in Table 5 below.

[0086] Table 5

[0087]

[0088]

[0089] PCR reaction conditions: 94℃ pre-denaturation for 5 min; followed by 32 cycles, each cycle consisting of: 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s; and a final extension at 72℃ for 10 min.

[0090] (2) Construction of recombinant expression plasmid pPICZαA-rLF-AIP

[0091] After the PCR product was completed, 8 μL of 6x DNA Loading was added. After mixing by pipetting, the sample was detected by 1% nucleic acid electrophoresis. The 501 bp band was excised and recovered using the Gel Extraction Kit D2500. The vector pPICZαA, which was double-digested with EcoRI and KpnI, was ligated overnight at 16°C. The ligation was then carried out by electroporation into DH5α competent cells and incubated at 37°C for 1 h on a shaker at 180 rpm. The cells were centrifuged and plated on LB solid medium containing bleomycin and incubated upside down at 37°C for 12 h. Single colonies were picked and cultured in Zeo-resistant LB liquid medium at 37°C for 6 h to obtain the recombinant strain DH5α-pPICZαA-rLF. After the bacterial culture became turbid, PCR was performed using the recombinant strain DH5α-pPICZαA-rLF-AIP as a template and the universal primers AOX1-F and AOX1-R for pPICZαA-rLF-AIP. The PCR system was basically the same as in Table 4, except that “pPICZαA-rLF genome” was replaced with an equal amount of “pPICZαA-rLF-AIP bacterial culture”. The PCR reaction conditions were the same as those described in the section “(1) Obtaining the target gene fragment rLF-AIP”. After PCR amplification, the recombinant plasmid gene was extracted and sent to bioengineering for sequencing. The positive plasmid was named the recombinant expression plasmid pPICZαA-rLF-AIP.

[0092] The gel electrophoresis results from PCR identification show that the target gene fragment of approximately 939 bp is consistent with expectations.

[0093] 1.5 Construction of recombinant strain KM71H-pPICZαA-rLF-AIP

[0094] (1) Plasmid linearization

[0095] The recombinant expression plasmid pPICZαA-rLF-AIP was linearized. The enzyme digestion system was as shown in Table 3, except that "pPICZαA-rLF" was replaced with an equal amount of "pPICZαA-rLF-AIP", while the other raw materials and amounts remained unchanged.

[0096] (2) Transformation into KM71H host bacteria

[0097] The KM71H strain was streaked onto YPD solid medium and incubated overnight. Single colonies were picked and cultured until OD500 was reached. 600 After washing twice with an appropriate amount of ddH2O, 100 μL of 1 mol / L sorbitol was added. The linearized recombinant plasmid was then added to KM71H competent cells and electroporated (1500 V, 4.8 ms). 1 mL of 1 mol / L sorbitol was added, and the mixture was incubated on ice for 5 min and then cultured at 28 °C and 200 rpm for 1 h. The supernatant was discarded by centrifugation and the mixture was plated on YPD solid medium to obtain the recombinant strain. The genome of the recombinant strain was extracted and subjected to PCR. The PCR system was basically the same as that in Table 4, except that “pPICZαA-rLF genome” was replaced with an equal amount of “pPICZαA-rLF-AIP genome”. The PCR reaction conditions were the same as those described in the section “(1) Obtaining the target gene fragment rLF-AIP”. After PCR identification and comparison, the strain containing the positive genome was the recombinant strain KM71H-pPICZαA-rLF-AIP.

[0098] Among them, the gel electrophoresis images of PCR identification are as follows: Figure 2 As shown. By Figure 2 As can be seen, a fragment of approximately 939bp was obtained, which is consistent with expectations.

[0099] Example 2

[0100] This embodiment provides a method for preparing the lactoferrin and anti-inflammatory peptide fusion protein provided in Example 1, the details of which are as follows:

[0101] 1. Protein expression

[0102] The recombinant strain KM71H-pPICZαA-rLF-AIP was cultured in YPD liquid medium containing Zeo, Kan, and Amp at 28°C for 24 h. The bacterial culture was then cultured in BMGY medium at 28°C until OD200. 600 The bacterial culture was centrifuged at 2500 rpm for 5 min, and the bacterial resuspended in BMMY medium. It was continuously cultured at 28℃ for 4 days, with methanol added every day. The supernatant was collected by centrifugation at 12000 rpm for 10 min, and 100% TCA was added. After mixing, the mixture was stored at -20℃. The supernatant was centrifuged at 12000 rpm for 20 min, and the protein precipitate was collected. The precipitate was dissolved in urea-free binding buffer and then sampled for SDS-PAGE analysis.

[0103] 2. Protein purification

[0104] The protein was placed in a prepared dialysis bag and dialyzed overnight at 4°C. The protein was then purified using a His-tagged protein purification kit to obtain a lactoferrin-anti-inflammatory peptide fusion protein (denoted as fusion protein rLF-AIP). The specific procedures are as follows:

[0105] a: Add Ni-Agarpse Resin packing to the chromatography column and let it stand for 10 min;

[0106] b: After the gel and solution separate into layers, open the bottom outlet to let the ethanol flow out;

[0107] c: All solutions used in the experiment should be filtered through a 0.45μm filter;

[0108] d: Add 5 column volumes of deionized water to the column to rinse away the ethanol;

[0109] e: Add 10 times the column volume of Binding Buffer to balance the column;

[0110] f: Load protein solution onto column at a flow rate of 10 times column volume per hour, and collect the first flow-through solution into a 50 mL tube;

[0111] j: The first flow-through fluid is loaded onto the column again;

[0112] h: Rinse the column with 15 times the column volume of Inclusion Body Binding Buffer;

[0113] i: Elute with 5 column volumes of Inclusion Body Elution Buffer and collect the eluent;

[0114] g: After elution, seal the column with 3 column volumes of Inclusion Body Binding Buffer and 5 column volumes of 20% anhydrous ethanol, and store at 4°C.

[0115] The recombinant strain KM71H-pPICZαA-rLF-AIP was induced and analyzed by SDS-PAGE and Western blot. The SDS-PAGE results of the recombinant strain KM71H-pPICZαA-rLF-AIP induced by SDS-PAGE are shown below. Figure 3 As shown in the figure. To better detect the fusion protein, this invention also induced the expression of recombinant strains KM71H-pPICZαA-rLF and KM71H-pPICZαA-rLF, and then purified the resulting protein to obtain the corresponding recombinant protein. A comparison of the Western blot detection results of recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP is shown in the figure. Figure 4 As shown, where Figure 4A indicates that after induction by the recombinant strain KM71H-pPICZαA-rLF, Figure 4 B indicates the recombinant strain KM71H-pPICZαA-rLF-AIP after induction.

[0116] Figure 3-4 This indicates that the recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP were successfully expressed, with protein sizes consistent with expectations and molecular weights of approximately 25 kDa and 33 kDa, respectively.

[0117] Comparative Example 1

[0118] This comparative example provides a recombinant bacterial strain and recombinant protein 28a-rLF constructed based on the prokaryotic expression vector pET-28a-rLF. The specific construction method is as follows:

[0119] 1. Obtaining the target gene

[0120] Based on the principles of enzyme digestion and ligation in PCR and molecular construction, primers for amplifying the target fragment on the vector were designed using Primer 6.0 software. 28a-rLF-F and 28a-rLF-R were used to amplify the target fragment of pET-28a-rLF. The universal primers T7 / T7t for pET-28a were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are shown in Table 6.

[0121] Table 6

[0122] Primer name Primer sequence Enzyme cleavage sites 28a-rLF-F 5'-ATGGGTCGCGGATCCGAATTCATGGGTTTTGTGTTTG-3' EcoRⅠ 28a-rLF-R 5'-CTCGAGTGCGGCCGCAAGCTTTCTACCCAAACCCAT-3' HindⅢ T7 5'-TAATACGACTCACTATAGGG-3' T7t 5'-GCTAGTTATTGCTCAGCGG-3'

[0123] Note: The underlined area is the restriction enzyme cleavage site.

[0124] Using recombinant plasmid pPICZαA-rLF as a template and 28a-rLF-F and 28a-rLF-R as primers, the target fragment of recombinant expression plasmid pET-28a-rLF was obtained by PCR. The PCR amplification system is shown in Table 5.

[0125] The PCR program is as follows: pre-denaturation at 98℃ for 3 min; followed by 32 cycles, each cycle consisting of: denaturation at 98℃ for 10 s, annealing at 55℃ for 10 s, extension at 72℃ for 20 s; and final extension at 72℃ for 10 min.

[0126] 2. Construction of recombinant expression plasmid pET-28a-rLF

[0127] After PCR, 8 μL of 6x DNA Loading was added to the product, and the mixture was mixed by pipetting. The product was then detected by 1% nucleic acid electrophoresis. The 423 bp band was excised from the gel and recovered using the Gel Extraction Kit D2500. The recovered gel product and pET28a vector were double-digested with EcoRI and HindIII. The digestion system is shown in Table 7 below.

[0128] Table 7

[0129] Reagent Name volume 10×CutSmart Buffer 5.0μL EcoRI 2.0μL HindIII 2.0μL pET28a / rLF 5.0μL <![CDATA[Sterilized ddH2O]]> 36.0μL Total volume 50.0μL

[0130] After double digestion, pET-28a and rLF were incubated overnight, and then recovered by gel extraction after detection by 1% agarose gel electrophoresis. The recovered samples were named pET-28a(E+H) and rLF(E+H), respectively, and ligated. The ligation system is shown in Table 8 below.

[0131] Table 8

[0132] reagents volume rLF(E+H) gene fragment 3.0μL pET28a(E+H) 5.0μL T4 DNA Ligase 1.0μL 10×T4 DNA Ligase Buffer 1.0μL Total volume 10.0μL

[0133] After overnight ligation at 16℃, transformation was performed using the same method as in the construction of the recombinant expression plasmid pPICZαA-rLF-AIP in Example 1. Using bacterial culture as a template, PCR identification was performed using the universal primers T7 and T7t of pET-28a. The PCR system is shown in Table 9 below.

[0134] Table 9

[0135] Reagent Name volume 2×Taq Mix 10.0μL pET-28a-rLF bacterial solution 2.0μL T7 1.0μL T7t 1.0μL <![CDATA[Sterilized ddH2O]]> 6.0μL Total volume 20.0μL

[0136] The recombinant plasmid gene was extracted and sent to Biosensor for sequencing. The positive plasmid was named recombinant expression plasmid pET-28a-rLF.

[0137] 3. Induced expression of recombinant proteins

[0138] The recombinant expression plasmid pET-28a-rLF was transformed into Shuffle T7-B competent cells. Single colonies were picked, and bacterial culture was performed for identification by PCR. The colonies were then sent to Sangon Biotech for sequencing to identify positive clones. The positive bacteria were named recombinant strain T7-B-pET-28a-rLF. The positive bacteria were cultured in LB / c liquid medium at 37°C until OD... 600 When the pH reaches 0.6–0.8, add 1 mmol / L IPTG for 4 h to induce induction.

[0139] 4. Solubility analysis of the expression product

[0140] Positive bacteria cultured in LB / Kan liquid medium at 37°C showed OD 600To a concentration of 0.6–0.8, add IPTG and induce at 30°C for 4 h. Centrifuge at 4°C to resuspend the bacterial cells, sonicate on ice for 15 min, centrifuge the bacterial solution at 4°C after sonication, collect 30 μL of supernatant and 30 μL of precipitate, add 10 μL of 4x protein loading buffer to prepare the sample, incubate in boiling water for 10 min, and perform SDS-PAEG electrophoresis to determine the form in which the protein is expressed.

[0141] After cleaning and leak testing the glass plates, fix them on the gel casting apparatus. Quickly add the separating gel and stacking gel between the glass plates according to Table 10, insert the comb, and after 10 minutes, remove the comb. Install the prepared gel into the vertical electrophoresis tank, add 1xSDS buffer solution, and add 10 μL of sample to each well for SDS-PAEG electrophoresis. Different preparation systems are shown in Table 10.

[0142] Table 10

[0143]

[0144] In this process, the recombinant expression plasmid pET-28a-rLF was transformed into the expression strain Shuffle T7-B and then induced. The bacterial cultures of the empty pET-28a vector strain and the recombinant pET-28a-rLF strain before and after induction were collected. The supernatant and precipitate were lysed and analyzed for SDS-PAGE solubility. The gel electrophoresis images are shown below. Figure 5 As shown. Figure 5 In the text, M represents the molecular weight standard of the protein; lane 1 represents pET-28a before empty-load induction; lane 2 represents pET-28a after empty-load induction; lane 3 represents pET-28a-rLF before induction; lane 4 represents pET-28a-rLF after induction; lane 5 represents the supernatant after pET-28a-rLF lysis; and lane 6 represents the precipitate after pET-28a-rLF lysis.

[0145] Depend on Figure 5 As can be seen, after induction, lanes 4-5 showed a target band at approximately 19.5 kDa, consistent with the expected size, proving successful expression of the recombinant protein. After sonication lysis, the protein was mainly expressed in the supernatant, indicating that the pET-28a-rLF protein is soluble.

[0146] 5. Purification of recombinant proteins

[0147] pET-28a-rLF protein was purified using a His-tagged protein purification kit to obtain purified recombinant protein 28a-rLF.

[0148] Comparative Example 2

[0149] This comparative example provides a recombinant strain and recombinant protein pYD1-rLF constructed based on the eukaryotic expression vector pYD1-rLF. The specific construction method is as follows:

[0150] 1. Obtaining the target gene

[0151] Using the recombinant expression plasmid pPICZαA-rLF as a template and pYD1-rLF-F and pYD1-rLF-R as primers, the target fragment of the recombinant expression plasmid pYD1-rLF was obtained by PCR. The PCR amplification system is shown in Table 11.

[0152] Table 11

[0153] Reagent Name volume 2×PrimeSTAR Max 25.0μL pYD1-rLF-F 2.0μL pYD1-rLF-R 2.0μL pPICZαA-rLF 2.0μL Sterilization ddH2O 19.0 Total volume 50.0μL

[0154] The PCR program is as follows: pre-denaturation at 98℃ for 3 min; followed by 32 cycles, each cycle consisting of: denaturation at 98℃ for 10 s, annealing at 55℃ for 10 s, extension at 72℃ for 30 s; and final extension at 72℃ for 10 min.

[0155] 2. Construction of recombinant expression plasmid pYD1-rLF

[0156] After PCR, 8 μL of 6x DNA Loading was added to the product, and the mixture was mixed by pipetting. The product was then detected by 1% nucleic acid electrophoresis. The 429 bp band was excised from the gel and recovered using the Gel Extraction Kit D2500. The recovered gel product and pYD1 vector were double-digested with NheⅠ and EcoRI. The digestion system is shown in Table 12 below.

[0157] Table 12

[0158]

[0159]

[0160] After double digestion with enzymes, pYD1 and rLF were incubated overnight, and then recovered by gel extraction after detection by 1% agarose gel electrophoresis. The recovered samples were named pYD1(E+N) and rLF(E+N), respectively, and ligated. The ligation system is shown in Table 13 below.

[0161] Table 13

[0162] reagents volume rLF(E+N) gene fragment 6.0μL pYD1(E+N) 2.0μL T4 DNA Ligase 1.0μL 10×T4 DNA Ligase Buffer 1.0μL Total volume 10.0μL

[0163] After overnight ligation at 16℃, transformation was performed using the same method as in the construction of the recombinant expression plasmid pPICZαA-rLF-AIP in Example 1. Using bacterial culture as a template, PCR identification was performed using the universal primers pYD1-F and pYD1-R for pYD1-rLF. The PCR system is shown in Table 14 below.

[0164] Table 14

[0165] Reagent Name volume 2×Taq Mix 10.0μL DH5α-pYD1-rLF bacterial liquid 2.0μL pYD1-F 1.0μL pYD1-N 1.0μL <![CDATA[Sterilized ddH2O]]> 6.0μL Total volume 20.0μL

[0166] The recombinant plasmid gene was extracted and sent to Biosensor for sequencing. The positive plasmid was named recombinant expression plasmid pYD1-rLF.

[0167] 3. Construction of recombinant strains and induction of recombinant protein expression

[0168] The recombinant expression plasmid pYD1-rLF was transformed into competent cells of strain EBY100. Single colonies were selected, and yeast genome was extracted using a bioengineered yeast genome extraction kit. PCR identification was performed using universal primers pYD1-F and pYD1-R, and the samples were then sent to bioengineered sequencing labs to identify positive clones. The positive strain was named recombinant strain EBY100-pYD1-rLF. Recombinant protein expression and purification were performed according to the method described in Example 2.

[0169] A recombinant strain EBY100-pYD1 with an empty vector pYD1 was constructed. Recombinant proteins from EBY100-pYD1 and EBY100-pYD1-rLF strains were induced, expressed, and purified, and detected by SDS-PAGE and Western blot. The SDS-PAGE results of EBY100-pYD1 and EBY100-pYD1-rLF strains after induction and purification are shown below. Figure 6 As shown. Figure 6 In the diagram, M represents the molecular weight standard of protein; lane 1 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1; lane 2 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1; lane 3 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1 after sonication; lane 4 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1 after sonication; lane 5 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1-rLF; lane 6 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF; lane 7 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication; and lane 8 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication. A comparison of the Western blot results of recombinant strains EBY100-pYD1 and EBY100-pYD1-rLF after induction culture and purification is shown in the figure below. Figure 7 As shown. Figure 7In the diagram, lane 1 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1, lane 2 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1, lane 3 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1 after sonication, lane 4 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1 after sonication, lane 5 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1-rLF, lane 6 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF, lane 7 represents the supernatant of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication, and lane 8 represents the precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication.

[0170] Figure 6-7 The results showed that the pYD1-rLF protein was successfully expressed, mainly in the precipitate and the precipitate after sonication, with a molecular weight of approximately 47.9 kDa, consistent with the expected size.

[0171] Example of effect 1

[0172] The present invention measures the in vitro antibacterial activity of different recombinant proteins involved in Example 1 and Comparative Examples 1-2, and the specific methods are as follows:

[0173] I. Determination of Antibacterial Activity of Eukaryotic Recombinant Proteins

[0174] Streaking of *Escherichia coli* and *Staphylococcus aureus* glycerol bacteria onto antibiotic-free LB agar plates and incubating at 37°C for 12 h. Single colonies were picked and cultured in antibiotic-free LB liquid medium for OD. 600 Adjust the culture medium to 0.6-0.8. After mixing the bacterial culture and LB solid medium and pouring the mixture into petri dishes to solidify, use a sterile punch with an inner diameter of approximately 8 mm to make holes. Details are as follows:

[0175] 1. Add 50, 100, 150, and 200 μL of supernatant from the recombinant strain KM71H-pPICZαA-rLF-AIP or KM71H-pPICZαA-rLF to each well, and incubate at 37°C for 12 h. Ampicillin was used as a positive control, and 200 μL of uninduced fermentation supernatant and empty-vector fermentation supernatant were used as negative controls. The antibacterial activity of the recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP was evaluated by measuring the diameter of the inhibition zone (mm).

[0176] The supernatant was collected by centrifugation at 12,000 rpm for 10 min, as described in the "1. Protein Expression" section of Example 2.

[0177] 2. Add 200 μL of recombinant strain EBY100-pYD1-rLF to each well, along with the supernatant and precipitate of the fermentation broth of recombinant strain EBY100-pYD1 or EBY100-pYD1-rLF, and the supernatant and precipitate of the fermentation broth of recombinant strain EBY100-pYD1-rLF after sonication. Incubate at 37℃ for 12 h. Ampicillin was used as a positive control, and strain pYD1 was used as a negative control. The antibacterial activity of pYD1-rLF expressed by Saccharomyces cerevisiae was evaluated by measuring the diameter (mm) of the inhibition zone. Specifically:

[0178] The positive strain was activated at 30°C for 24 hours in YPD liquid medium containing Zeocin at a ratio of 1:100, and then transferred to 100 mL of YNB-CAA medium at a ratio of 1:50 and cultured until OD. 600 The concentration was 0.8-1.2 (the culture medium became turbid). After centrifugation to collect the bacterial cells, they were resuspended in 100 mL of YNB-CAA medium for induction expression at 25°C for 72 h. After induction, the bacterial cells were collected by centrifugation, resuspended in 10 mL of pre-cooled PBS, and then sonicated on ice (300 W, 3 seconds per sonication, 5-second interval, total duration 15 minutes). The lysis buffer was centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant (soluble fraction) and precipitate (insoluble fraction resuspended in PBS) were collected separately. 30 μL of each sample was added to 10 μL of 4× loading buffer, denatured in a boiling water bath for 10 minutes, and then subjected to SDS-PAGE electrophoresis to analyze the expression form and solubility of the target protein.

[0179] II. Determination of Antibacterial Activity of Prokaryotic Recombinant Protein: The same concentration of recombinant protein pET-28a-rLF was added to each well and incubated at 37°C for 12 h. Ampicillin was used as a positive control. The antibacterial activity of the prokaryotic recombinant protein pET28a-rLF was evaluated by measuring the diameter (mm) of the inhibition zone.

[0180] The preparation method of the fermentation supernatant of pET-28a-rLF is as follows:

[0181] The recombinant strain T7-B-pET-28a-rLF was inoculated into 100 mL of LB / Kan liquid medium and cultured at 37 °C with shaking at 220 rpm until OD500 was reached. 600The concentration was 0.6–0.8. Subsequently, 100 μL of 1M IPTG (final induction concentration 1 mmol / L) was added to the culture, and expression was induced at 30°C and 220 rpm for 4 hours. After induction, the bacterial cells were collected by centrifugation at 4000 rpm for 20 minutes at 4°C. The supernatant was discarded, and the cells were thoroughly resuspended in 10 mL of pre-chilled PBS buffer. The resuspended bacterial solution was placed in an ice-water mixture and subjected to sonication on ice (300 W, 3 seconds of sonication followed by a 5-second interval, for a total of 15 minutes). The lysate was centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant (soluble components) and precipitate (inclusion bodies and insoluble components) were carefully separated.

[0182] III. Results of Antibacterial Activity Assay

[0183] The results of the antibacterial activity assays of recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP against Escherichia coli ETEV K88 and Staphylococcus aureus are shown in Table 15. Figure 8 As shown. Figure 8 middle Figure 8 A represents the recombinant protein pPICZαA-rLF. Figure 8 B represents the recombinant protein pPICZαA-rLF-AIP; the correspondence between the wells labeled in the figure and the added substances is as follows: 1. Empty fermentation supernatant; 2. Ampicillin; 3. 200 μL uninduced supernatant; 4–7.50, 100, 150, and 200 μL supernatant. The antibacterial activity of pYD1-rLF expressed by *Saccharomyces cerevisiae* EBY100 against *Escherichia coli* ETEV K88 and *Staphylococcus aureus* is shown in the figure. Figure 9 As shown. Figure 9 In the table, the correspondence between wells with different labels and the added substances is as follows: 1. pYD1 whole bacteria; 2. pYD1-rLF whole bacteria; 3-6. pYD1-rLF supernatant, precipitate, supernatant after sonication, and precipitate after sonication. The antibacterial activity of recombinant protein pET28a-rLF against *Escherichia coli* ETEV K88 and *Staphylococcus aureus* is shown in Table 16. Figure 10 As shown. Figure 10 middle, Figure 10 A represents the recombinant protein pET28a-rLF. Figure 10 B is the negative control.

[0184] Table 15

[0185]

[0186] Table 16

[0187]

[0188]

[0189] Depend on Figure 8-10 As shown in Table 15, no inhibition zone was observed around the recombinant protein pYD1-rLF; an inhibition zone was observed around the recombinant protein pET28a-rLF; inhibition zones were observed around the recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP, and the diameter of the inhibition zone varied according to the volume of the supernatant. No inhibition zones were observed around the supernatant of uninduced and empty-carrier fermentation. In summary, the inhibition zone method shows that the recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP can be secreted into the supernatant and have antibacterial activity against both Escherichia coli and Staphylococcus aureus, with better inhibitory activity against Escherichia coli than against Staphylococcus aureus. The antibacterial activity of the eukaryotic protein was better than that of the prokaryotic protein.

[0190] Example 2

[0191] This invention measures the antibacterial activity of different recombinant proteins involved in Example 1 and Comparative Examples 1-2 in mice. The specific methods are as follows:

[0192] I. Animal Immunization Experiments

[0193] Seventy 4-week-old female BALB / c mice were randomly divided into seven groups: a saline control group (NC group), a no-load control group, a recombinant protein pPICZαA-rLF control group (F group), a recombinant protein pPICZαA-rLF-AIP control group (FA group), an ETEC K88 infection group (K88 group), a recombinant protein pPICZαA-rLF treatment group (F+K88 group), and a recombinant protein pPICZαA-rLF-AIP treatment group (FA+K88 group). Mice were orally administered recombinant protein, 2 mg / mouse, once daily for 28 days. On day 27, the control group received an intraperitoneal injection of saline, while the infection and treatment groups received an intraperitoneal injection of ETEC K88 (0.5 mL, 2 × 10⁻⁶ mg / mcg). 9 (CFU / mL), and the grouping details are shown in Table 17.

[0194] Table 17

[0195]

[0196] After being challenged with K88, mice in the K88 group exhibited dull fur, huddled together with their eyes closed, reduced activity and food intake, decreased sensitivity to external stimuli, and sluggish responses. The clinical symptoms in the treatment group were milder than those in the K88 challenge group. Mice in the control group had smooth, glossy fur, normal activity and appetite, and showed no clinical symptoms.

[0197] To further determine the infection status of the mice, necropsies were performed on mice that were already dead, critically ill and near death, or had lost more than 20% of their body weight. The overall pathological changes in the infected mice are shown in the following figure. Figure 11 As shown. By Figure 11 It was found that after dissection, the intestines and liver were edematous and congested. The treatment group had milder symptoms than the virus-infected group, while the control group showed normal symptoms and no obvious pathological symptoms.

[0198] Mice that were already dead, critically ill and about to die, or had lost more than 20% of their body weight were dissected, and samples were collected for histopathological evaluation of the intestines, liver, spleen, and kidneys. The pathological changes of the intestines, liver, spleen, and kidneys in mice from different groups are shown in the following figures. Figure 12 As shown. Typical gross pathological changes in the four important organs—intestine, liver, spleen, and kidney—include intestinal congestion and enlargement; liver enlargement and congestion with blunt, thickened edges and a fragile, easily ruptured texture; spleen enlargement, roundness, congestion, and a dark red color; and kidney enlargement and congestion. Figure 12 It can be seen that the lesions in the intestine, liver, spleen and kidney of the mice in the treatment group were milder than those in the challenge group, while no obvious lesions were found in the control group.

[0199] II. Evaluation Indicators and Results

[0200] 1. Assessment of histopathological changes

[0201] HE staining was used to further examine the histopathological examination of mouse tissues, including the intestines, liver, spleen, and kidneys, to investigate pathological damage. HE staining images of the intestines, liver, spleen, and kidneys from different groups of mice are shown below. Figure 13 As shown.

[0202] Depend on Figure 13It was found that histopathological damage was observed in the intestines, liver, spleen, and kidneys of mice in the challenge control group (i.e., the K88 group): the intestinal mucosal layer structure was destroyed, the villi were severely atrophied and degenerated, and a large number of intestinal villi epithelial and intestinal gland epithelial cells were sloughed off (red arrows). Sloughed intestinal villi and epithelial cells were visible in the intestinal lumen. A large number of intestinal villi epithelial cells were separated from the lamina propria (blue arrows), with widened gaps and loose arrangement. Muscle cells were unevenly stained. Vacuole degeneration (blue arrows) and vascular congestion (red arrows) were observed in the liver. Extensive hemorrhage (red arrows) and a small number of granulocyte infiltrations (purple arrows) were observed in the spleen. In the kidney tissue, occasional dilation of renal capsules (light green arrows), hydropic degeneration of renal tubular epithelial cells (green arrows), necrosis of a small number of renal tubular epithelial cells (black arrows), sloughed epithelial cells visible in a small number of renal tubular lumens (light blue arrows), occasional infiltration of granulocytes and lymphocytes (purple arrows), and a small number of interstitial vascular congestions (red arrows) were observed.

[0203] In contrast, the NC, empty vector, F, and FA groups, which were not challenged with the virus, showed normal morphology and regular arrangement of their organs, with no obvious inflammatory cell infiltration or abnormalities. No obvious inflammatory cell infiltration was found in the recombinant protein pPICZαA-rLF and recombinant protein pPICZαA-rLF-AIP treatment groups, but mild damage was observed: short and thick intestinal villi, and a small amount of intestinal villi epithelial and intestinal gland epithelial cells detached (red arrows); occasional hepatic vascular congestion (red arrows); a small amount of splenic sinusoidal congestion (red arrows); and a small amount of renal interstitial vascular congestion (red arrows). These pathological section results further demonstrate that recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP can effectively prevent ETEC K88 from causing pathogenic damage to tissues, providing effective protection against ETEC K88 infection.

[0204] 2. Escherichia coli count test

[0205] The number of *E. coli* in the mesentery, liver, spleen, and cecum of mice was determined and counted using the dilution plate count method. The tissues were homogenized and serially diluted (10⁻⁶ oz). -4 -10 -6 Each diluted suspension (50 μL) was inoculated onto selective culture plates and incubated at 37°C for 18 h. Escherichia coli was then counted using the plate count method, with each group repeated three times. No statistically significant difference in E. coli load was observed in the tissues of the uninfected groups (NC, empty vector, F, and FA). Therefore, this invention analyzes the data from the infected groups.

[0206] The statistical results of bacterial load in the mesentery, liver, spleen, and cecum of mice in different groups are as follows: Figure 14 As shown. Among them, Figure 14A represents the statistical results of Escherichia coli load in the mesentery of the K88 group, F+K88 group, and FA+K88 group. Figure 14 B represents the statistical results of Escherichia coli load in the livers of the K88 group, F+K88 group, and FA+K88 group. Figure 14 C represents the statistical results of Escherichia coli load in the spleen of the K88 group, F+K88 group, and FA+K88 group. Figure 14 D represents the statistical results of Escherichia coli load in the cecum of the K88 group, F+K88 group, and FA+K88 group.

[0207] Depend on Figure 14 Compared with the K88 infection group, the E. coli load in the mesentery of the recombinant protein pPICZαA-rLF treatment group was significantly reduced (P < 0.05), and the E. coli load in the mesentery of the recombinant protein pPICZαA-rLF-AIP treatment group was extremely significantly reduced (P < 0.01). Compared with the K88 infection group, the number of E. coli in the liver of mice treated with recombinant protein pPICZαA-rLF was significantly reduced (P < 0.05), and the number of E. coli in the liver of mice treated with recombinant protein pPICZαA-rLF was also reduced. Compared with the K88 infection group, both recombinant protein pPICZαA-rLF and pPICZαA-rLF-AIP treatment groups reduced the number of E. coli in the spleen and cecum. In conclusion, recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP can significantly reduce the number of E. coli in the mesentery and liver of mice, and reduce the number of E. coli in the spleen and cecum. This indicates that the two recombinant proteins can prevent E. coli from migrating from the abdomen to nearby organs.

[0208] 3. Measurement and results of the concentrations of cytokines TNF-α, IL-1β, IFN-γ, IL-10 and sIgA

[0209] The levels of serum cytokines TNF-α, IL-1β, IFN-γ, IL-10, and sIgA secreted from the terminal ileum in mice were measured using a Jiangsu enzyme immunoassay kit. Statistical results of cytokines in the serum of mice from different groups are shown below. Figure 15 As shown; where, Figure 15 A represents the statistical results of serum levels of the pro-inflammatory factor TNF-α in mice from different groups. Figure 15 B represents the statistical results of serum levels of the pro-inflammatory factor IL-1β in mice from different groups. Figure 15 C represents the statistical results of IFN-γ levels in the serum of mice from different groups. Figure 15 D represents the statistical results of the anti-inflammatory factor IL-10 in the serum of mice in different groups. Figure 15 E represents the statistical results of sIgA, a mucosal defense molecule, in the serum of mice from different groups.

[0210] Depend on Figure 15 The results showed that the concentration of the pro-inflammatory cytokine TNF-α in the K88 group was lower than that in other groups. Recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP could reduce TNF-α concentration, with pPICZαA-rLF showing a significant effect (P < 0.05). Compared with the K88 group, recombinant proteins pPICZαA-rLF and pPICZαA-rLF-AIP reduced the concentration of the pro-inflammatory cytokine IL-1β in serum, with pPICZαA-rLF-AIP showing a highly significant difference (P < 0.001). Compared with the K88 group, both recombinant proteins reduced the concentration of IFN-γ in serum. Compared with the K88 group, both recombinant proteins increased the concentration of the anti-inflammatory cytokine IL-10 in serum, with pPICZαA-rLF-AIP showing a significant effect (P < 0.05). The concentration of sIgA in the terminal ileum of the K88 group was higher than that in other groups. The two recombinant proteins reduced the concentration of sIgA, with the recombinant protein pPICZαA-rLF-AIP showing the most significant effect (P < 0.05).

[0211] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lactoferrin-anti-inflammatory peptide fusion protein, characterized in that: It has the amino acid sequence shown in SEQ ID NO:

1.

2. A recombinant expression vector, characterized in that: It can express the lactoferrin and anti-inflammatory peptide fusion protein as described in claim 1.

3. The recombinant expression vector as described in claim 2, characterized in that: The recombinant expression vector uses a eukaryotic expression vector as its backbone.

4. A recombinant bacterial strain, characterized in that: It includes the recombinant expression vector as described in claim 2 or 3.

5. The recombinant strain according to claim 4, characterized in that: The recombinant strain used yeast as the starting strain.

6. The use of the lactoferrin and anti-inflammatory peptide fusion protein according to claim 1 in the preparation of antibacterial drugs.

7. The application of the lactoferrin and anti-inflammatory peptide fusion protein as described in claim 6 in the preparation of antibacterial drugs, characterized in that: The antibacterial drug includes at least one of the following: a drug that inhibits Gram-negative bacteria, a drug that inhibits Gram-positive bacteria, or a drug that simultaneously inhibits both Gram-negative and Gram-positive bacteria.

8. The application of the lactoferrin and anti-inflammatory peptide fusion protein as described in claim 7 in the preparation of antibacterial drugs, characterized in that: The Gram-negative bacteria include Escherichia coli; and / or The Gram-positive bacteria include Staphylococcus aureus.

9. The use of the recombinant expression vector according to claim 2 or 3 in the preparation of antibacterial drugs.

10. The use of the recombinant strain according to claim 4 or 5 in the preparation of antibacterial drugs.

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