A complex preparation containing bovine lactoferrin and lyticase and its use

Abstract

The application discloses a complex preparation containing bovine lactoferrin and lyticase and application thereof, and belongs to the technical field of biology. The preparation is composed of bovine lactoferrin and streptococcal lyticase, and both are prepared through a baculovirus-insect cell eukaryotic expression system. Researches show that the complex preparation has a significant synergistic antibacterial effect. The complex preparation not only can effectively inhibit a plurality of gram-positive bacteria, but also can overcome the limitation that single streptococcal lyticase has weak activity on gram-negative bacteria, and significantly enhances the inhibiting effect on escherichia coli and salmonella. Especially, the complex preparation still has clear in-vitro antibacterial activity on multi-drug resistant escherichia coli such as ciprofloxacin and doxycycline, thereby providing a new way for developing a new type of drug for resisting drug-resistant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a compound preparation containing bovine lactoferrin and lyase and its application. Background Technology

[0002] The irrational use of antibiotics has led to the rapid spread of multidrug-resistant (MDR) bacteria, posing a serious challenge to global public health. To address this issue, the scientific community has extensively explored various antibiotic alternatives, many of which have already been commercialized. Currently, probiotic preparations, antimicrobial lipopeptides, plant extracts, plant essential oils, acidifiers, and xylooligosaccharides are widely used in livestock and poultry farming to replace antibiotics. However, the quality of these "antibiotic alternatives" on the market varies greatly, and their actual antibacterial effects are often unsatisfactory, failing to fully meet the needs of practical production.

[0003] Bovine lactoferrin is a natural iron-binding glycoprotein. As a novel antibacterial agent, it possesses advantages such as broad-spectrum antibacterial activity, safety, non-toxicity, and natural origin. In addition to its antibacterial effects, it also exhibits various biological functions including antiviral, anti-inflammatory, anticancer, and immunomodulatory effects, attracting widespread attention in recent years. However, currently, bovine lactoferrin is mostly produced through prokaryotic expression systems, which often encounter problems such as protein folding errors and inclusion body formation, leading to difficulties in subsequent purification and low yields, thus limiting its large-scale application.

[0004] Bacteriophage lysins exhibit strong lytic activity against Gram-positive bacteria, but their effectiveness against Gram-negative bacteria is significantly limited by the presence of the outer membrane barrier. They typically require combination with membrane-penetrating agents or modification through genetic engineering to achieve their full efficacy. In my country's livestock and poultry farming, streptococcal-specific lysins have been used for the control of pathogens such as streptococci and Staphylococcus aureus, but their activity against Gram-negative bacteria is weak, and their antibacterial spectrum is narrow, hindering their widespread application. Therefore, effectively improving the antibacterial efficacy of bacteriophage lysins, especially expanding their activity against Gram-negative bacteria, has become a crucial issue that urgently needs to be addressed.

[0005] It is worth noting that no studies have yet reported a strategy of combining bovine lactoferrin with bacteriophage lysins to enhance the latter's antibacterial effect. Complementing the advantages of both may provide new ideas and approaches for developing highly effective and broad-spectrum novel antibacterial agents. Summary of the Invention

[0006] This invention provides a compound formulation containing bovine lactoferrin and lyase, and its application in the development of new antibacterial agents.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] First, the present invention provides a compound antibacterial preparation containing bovine lactoferrin and streptococcal lysin, wherein the compound antibacterial preparation is composed of bovine lactoferrin and streptococcal lysin, wherein the concentration of bovine lactoferrin is 2000 μg / mL and the concentration of streptococcal lysin is 512 μg / mL.

[0009] Preferably, the bovine lactoferrin is a protein obtained by cloning the bovine lactoferrin gene sequence SEQ ID NO.2 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system;

[0010] The streptococcal lysin is a protein obtained by cloning the streptococcal lysin gene sequence SEQ ID NO.1 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system.

[0011] Preferably, the antibacterial spectrum of the compound antibacterial preparation includes Staphylococcus aureus, Staphylococcus discoloration, Staphylococcus epidermidis, cecal cocci, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, Escherichia coli, and Salmonella.

[0012] Preferably, the antibacterial agent has a MIC of 16 μg / mL against Staphylococcus aureus, a MIC of 250 μg / mL against Escherichia coli, a MIC of 250 μg / mL against Salmonella, and a MIC of 32 μg / mL against Streptococcus suis.

[0013] Secondly, the present invention provides the application of a composition containing bovine lactoferrin and streptococcal lysin in the preparation of an antibacterial agent, wherein the concentration of bovine lactoferrin in the composition is 2000 μg / mL and the concentration of streptococcal lysin is 512 μg / mL.

[0014] Preferably, the bovine lactoferrin is a protein obtained by cloning the bovine lactoferrin gene sequence SEQ ID NO.2 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system;

[0015] The streptococcal lysin is a protein obtained by cloning the streptococcal lysin gene sequence SEQ ID NO.1 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system.

[0016] Preferably, the antibacterial agent has an antibacterial spectrum including Staphylococcus aureus, Staphylococcus discoloration, Staphylococcus epidermidis, cecal cocci, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, Escherichia coli, and Salmonella.

[0017] Finally, the present invention provides the use of a composition of bovine lactoferrin and streptococcal lysin in the preparation of a medicament for inhibiting drug-resistant Escherichia coli, wherein the concentration of bovine lactoferrin is 2000 μg / mL and the concentration of streptococcal lysin is 512 μg / mL.

[0018] Preferably, the bovine lactoferrin is a protein obtained by cloning the bovine lactoferrin gene sequence SEQ ID NO.2 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system;

[0019] The streptococcal lysin is a protein obtained by cloning the streptococcal lysin gene sequence SEQ ID NO.1 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system.

[0020] Preferably, the drug-resistant bacteria are Escherichia coli isolates that are resistant to ciprofloxacin, doxycycline, gentamicin, enrofloxacin, and spectinomycin, but only have low sensitivity to cefixime.

[0021] The beneficial effects of this invention are as follows:

[0022] First, this invention combines bovine lactoferrin and streptococcal lysin, achieving complementary advantages and synergistic effects between the two components. Streptococcal lysin alone has almost no inhibitory effect on Gram-negative bacteria (such as Escherichia coli and Salmonella); while bovine lactoferrin alone has limited inhibitory effect on Gram-positive bacteria (such as Staphylococcus aureus and Streptococcus suis). However, when the two are combined, they not only effectively inhibit a variety of Gram-positive bacteria but also significantly enhance the inhibitory effect on Gram-negative bacteria, achieving broad-spectrum inhibition against a variety of common pathogens and overcoming the limitation of narrow antibacterial spectrum of single components.

[0023] Secondly, the compound formulation of this invention not only broadens the antibacterial spectrum but also significantly improves the antibacterial efficiency. Taking Staphylococcus aureus as an example, a single streptococcal lysin requires 12 hours to completely kill the bacteria, while the compound formulation only requires 4 hours; for Escherichia coli, a single bovine lactoferrin requires 12 hours to completely kill the bacteria, while the compound formulation completes the process within 8 hours, greatly improving the antibacterial speed and enabling faster control of pathogen infection.

[0024] Furthermore, facing the increasingly severe challenge of drug-resistant bacteria, the compound antibacterial formulation of this invention demonstrates great potential. In in vitro experiments, neither bovine lactoferrin nor streptococcal lysin alone could inhibit multidrug-resistant Escherichia coli, but the compound formulation produced a clear inhibition zone. This indicates that, through ingenious combination, this invention provides an effective alternative for developing novel drugs against drug-resistant bacteria.

[0025] Finally, both bovine lactoferrin and streptococcal lyase of the present invention are produced by a baculovirus-insect cell eukaryotic expression system. This system can ensure the correct folding and post-translational modification of proteins, resulting in proteins with high purity, strong activity, and good safety. It avoids the problems of inclusion bodies and purification difficulties that are easy to generate in prokaryotic expression systems, and is more conducive to large-scale production and application. Attached Figure Description

[0026] Figure 1 Results of indirect immunofluorescence assay for recombinant baculovirus packaging

[0027] (a) Immunofluorescence image of recombinant baculovirus with streptococcal lysin; (b) Immunofluorescence image of recombinant baculovirus with bovine lactoferrin; (c) Image of the negative control group;

[0028] Figure 2 SDS-PAGE and Western Blot analysis of bovine lactoferrin;

[0029] Figure 3 SDS-PAGE and Western Blot analysis of streptococcal lysin;

[0030] Figure 4 The purification results are for streptococcal lysin and bovine lactoferrin.

[0031] Figure 5 The antibacterial effect of streptococcal lysin on Gram-positive bacteria;

[0032] Figure 6 The antibacterial effect of streptococcal lysin on streptococci;

[0033] Figure 7 The antibacterial effect of bovine lactoferrin against Escherichia coli (a) and Salmonella (b);

[0034] Figure 8 For drug resistance testing of drug-resistant Escherichia coli;

[0035] Figure 9 The effect of the compound preparation on the antibacterial activity of drug-resistant Escherichia coli. Detailed Implementation

[0036] Example 1

[0037] Preparation of recombinant bovine lactoferrin and streptococcal lysin

[0038] 1.1 Gene Construction and Recombinant Plasmid Synthesis

[0039] (1) Based on the phage lysin Plyss2 gene sequence and the bovine lactoferrin gene sequence, the target gene sequence of Plyss2 gene (SEQ ID NO.1) and the bovine lactoferrin gene sequence (SEQ ID NO.2) were constructed by gene modification and optimization.

[0040] (2) Using the baculovirus-insect cell eukaryotic expression system, the optimized gene was cloned into the pFastBac1 vector. The specific steps for synthesizing the recombinant plasmid are as follows:

[0041] The plasmid carrying streptococcal lysin was amplified by PCR using the corresponding primers (primer sequences shown in SEQ ID NO.3 and SEQ ID NO.4), and the target fragment was recovered by gel electrophoresis. The target protein gene and the pFastbac-1 vector were double-digested with BamHI and EcoRI, respectively, to obtain the target fragment and vector with the same sticky ends, which were then recovered by gel electrophoresis. The recovered target gene and pFastbac-1 vector were ligated using T4 DNA ligase to obtain a recombinant plasmid carrying streptococcal lysin.

[0042] The plasmid carrying bovine lactoferrin was amplified by PCR using the corresponding primers (primer sequences shown in SEQ ID NO.5 and SEQ ID NO.6). The target fragment was recovered by gel electrophoresis. The target protein gene and the pFastbac-1 vector were digested with SalⅠ and NotⅠ, respectively, to obtain the target fragment and vector with the same sticky ends. The recovered target gene and pFastbac-1 vector were ligated using T4 DNA ligase to obtain the recombinant plasmid carrying bovine lactoferrin.

[0043] The recombinant plasmids described above were transformed into Escherichia coli DH5α competent cells and cultured at 37°C and 200 rpm for 12 hours with constant temperature shaking.

[0044] By using blue-white screening, white monoclonal colonies were selected, recombinant bacterial cells were extracted, and PCR identification was performed to screen for recombinant bacterial cells containing the target gene.

[0045] After the recombinant rod-shaped particles were completely dissolved, they were identified by PCR. The primers were designed as follows:

[0046]

[0047] Note: The product size is approximately the size of the inserted fragment + 2300bp.

[0048] 1.2 Packaging of recombinant baculovirus

[0049] The selected recombinant baculoviruses were transfected into SF9 adherent cells using transfection reagents to package the recombinant baculoviruses.

[0050] After 72 hours of culture, the cell supernatant was collected, and the successful packaging of the recombinant baculovirus was verified by indirect immunofluorescence assay.

[0051] The results of the immunofluorescence assay are as follows Figure 1 As shown, from Figure 1 As can be seen, (a) the streptococcal lysin recombinant baculovirus and (b) the bovine lactoferrin recombinant baculovirus have obvious immunofluorescence, while (c) the negative control group has no immunofluorescence, indicating that the streptococcal lysin recombinant baculovirus and bovine lactoferrin recombinant baculovirus of the present invention were successfully packaged.

[0052] 1.3 Protein Expression and Validation

[0053] SF9 suspension cells were infected with successfully packaged recombinant baculovirus for in vitro protein expression under the following conditions: 27°C and 120 rpm.

[0054] After culturing for 96 hours, the cell supernatant was collected by centrifugation, and the expression of bovine lactoferrin and streptococcal lysin was verified by SDS-PAGE and Western Blot.

[0055] The results obtained are as follows Figure 2 and Figure 3 As shown.

[0056] from Figure 2 and Figure 3 The results show that bovine lactoferrin has a molecular weight of approximately 80 kDa, and streptococcal lyase has a molecular weight of approximately 35 kDa, and both were successfully expressed.

[0057] 1.4 Scale-up preparation and purification

[0058] The optimal expression conditions were obtained by optimizing the multiple of infection (MOI) of recombinant baculovirus infection of SF9 suspension cells and the sample collection time: MOI=5, sample collection time 96 hours.

[0059] Scale-up expression was carried out using a fully suspended cell bioreactor with a culture volume of 10 L.

[0060] The recombinant proteins were purified by affinity chromatography with a purity of over 95%. After ultrafiltration and concentration, bovine lactoferrin at a concentration of 2.4 mg / mL and streptococcal lyase at a concentration of 1 mg / mL were obtained, respectively.

[0061] The results are shown in Figure 4. Figure 4 It can be seen that the recombinant protein of streptococcal lysin and the recombinant protein of bovine lactoferrin were successfully purified.

[0062] Example 2

[0063] Detection of the antibacterial effect of recombinant streptococcal lysin protein against Gram-positive bacteria

[0064] (1) Prepare TSA culture plates for the resuscitation of the following strains:

[0065] 26 streptococcal strains (including 24 strains of Streptococcus suis, 1 strain of Streptococcus agalactiae, and 1 strain of Streptococcus dysgalactiae).

[0066] Staphylococcus aureus, Staphylococcus discolor, Staphylococcus epidermidis

[0067] 1 strain of cecal cocci

[0068] Escherichia coli, Salmonella, Pasteurella, Actinobacillus pleuropneumoniae

[0069] Each strain was streaked onto the corresponding culture medium plate and incubated in a 37°C biochemical incubator for 24 hours.

[0070] (2) The next day, pick a single colony from each plate and inoculate it into an appropriate liquid culture medium. Place it in a 37°C biochemical incubator and incubate for 24 hours.

[0071] (3) Prepare culture medium plates in advance and dry them overnight in an incubator at 37°C. Use a pipette to draw 100 μL of bacterial solution and spread it evenly on the surface of the culture medium plate. Spread it evenly with a sterile spreader and then let it air dry.

[0072] (4) Take 2 μL of protein sample stock solution and spot it onto the surface of the plate (avoid contact between the pipette tip and the culture medium, and replace the pipette tip after each spotting to prevent contamination). Place the plate upright and wait for the sample to be completely absorbed before inverting it and incubating it in a 37°C incubator for 24 hours.

[0073] (5) Observe and record the results after the event (e.g.) Figure 5 (3 Staphylococcus aureus isolates - 1 / 3 / 4, 1 Staphylococcus discoloration, 1 Staphylococcus epidermidis, and 1 Cecal coccus) and Figure 6 As shown (1-26 are streptococci: 24 strains of Streptococcus suis (except for numbers 13 and 16), 1 strain of Streptococcus agalactiae (number 16), 1 strain of Streptococcus dysgalactiae (number 13); 1 strain of Streptococcus lactis)).

[0074] from Figure 5 The results show that the recombinant streptococcal lysin protein prepared in this invention has significant antibacterial activity against clinical isolates of Staphylococcus aureus, Staphylococcus vera, Staphylococcus epidermidis, and Enterococcus cecum, with inhibition zones between 8-10 mm. However, it has no significant antibacterial activity against clinical isolates of Escherichia coli, Salmonella, Pasteurella, and Actinobacillus pleuropneumoniae.

[0075] At the same time, from Figure 6The results show that the recombinant streptococcal lysin protein prepared in this invention has a significant inhibitory effect on 23 strains of Streptococcus suis, 1 strain of Streptococcus agalactiae, 1 strain of Streptococcus dysgalactiae, and 1 strain of Streptococcus lactis.

[0076] Example 3

[0077] The MIC of recombinant streptococcal lysin protein against susceptible bacteria was determined.

[0078] (1) Take a 96-well cell culture plate and add 100 μL of the corresponding broth to each well (96 wells in total) using a 100 μL pipette. Add 100 μL of recombinant streptococcal lysin protein solution with an initial concentration of 512 μg / mL to the first well of each column in rows 1-8 (A1, B1, C1, D1, E1, F1, G1, H1).

[0079] (2) Using a pipette, draw 100 μL of liquid from the first well (A1-H1) of each row (AH) and transfer it to the second well. Mix thoroughly, then draw 100 μL from the second well and transfer it to the third well, and so on, serially diluting up to the tenth well. Discard 100 μL of liquid from the tenth well. Do not add drug solution to column 11 (positive control, containing only bacterial culture) and column 12 (negative control, without sterile culture or drug).

[0080] (3) Add 10 μL of pre-prepared 1×10^6 CFU / mL bacterial solution to each well in columns 1 to 11, mix gently, so that the final bacterial solution concentration in each well is about 1×10^5 CFU / mL, and do not add bacterial solution to column 12.

[0081] (4) Seal the 96-well plate with sealing film and incubate it in air at 37°C for 18±2 hours. Results can only be read when sufficient bacterial growth is shown in the positive control well (column 11). Compare the bacterial growth in each well with that in the positive control well to determine the lowest drug concentration that can significantly inhibit bacterial growth, i.e., the minimum inhibitory concentration (MIC). The results are shown in Table 1.

[0082] Table 1. MICs of recombinant streptococcal lysin proteins against different bacteria.

[0083]

[0084] The results in Table 1 show that the recombinant streptococcal lysin protein exhibits antibacterial activity against Staphylococcus aureus, Streptococcus suis, and Enterococcus cecum. The inhibitory effect against Staphylococcus aureus is the strongest (MIC = 32 μg / mL), while the inhibitory effects against Streptococcus suis and Enterococcus cecum are weaker (MIC = 64 μg / mL). These results suggest that this recombinant protein may have greater application potential against Staphylococcus aureus infections, while its antibacterial effects against Streptococcus suis and Enterococcus cecum may require further optimization or combination with other antibacterial agents.

[0085] Example 4

[0086] Detecting the inhibitory effect of bovine lactoferrin recombinant protein on Gram-negative bacteria

[0087] (1) Prepare TSA or NA medium plates for the resuscitation of Salmonella and Escherichia coli strains. Streak each strain onto the corresponding medium plate and incubate at 37°C for 24 hours.

[0088] (2) The next day, pick a single colony from each plate and inoculate it into an appropriate liquid culture medium (such as LB medium). Place it in a 37°C biochemical incubator and incubate for 24 hours to prepare the bacterial culture.

[0089] (3) Measure the OD600 value of the bacterial culture and dilute it with sterile culture medium to about 1×10^6 CFU / mL for later use.

[0090] (4) Prepare TSA culture medium plates in advance and dry them overnight in an incubator at 37°C. Use a pipette to draw 100 μL of the adjusted bacterial solution (1×10^6 CFU / mL), spread it evenly on the surface of the culture medium plate, spread it evenly with a sterile spreader, and then let it air dry.

[0091] (5) Take 2 μL of bovine lactoferrin recombinant protein stock solution and spot it onto the surface of the plate (avoid contact between the pipette tip and the culture medium, and replace the pipette tip after each spotting to prevent contamination). Place the plate upright and, after the sample is completely absorbed, invert it and place it in a 37°C incubator for 24 hours.

[0092] (6) Observe the formation of inhibition zones on the plates, measure the diameter of the inhibition zones (unit: mm), and record the inhibitory effects on Salmonella and Escherichia coli. The results are as follows: Figure 7 As shown.

[0093] from Figure 7 The results showed that bovine lactoferrin recombinant protein (protein concentration of 2.4 mg / mL) had a significant antibacterial effect against Gram-negative bacteria, with inhibition zones of 10 mm and 12 mm against Salmonella and Escherichia coli, respectively.

[0094] Example 5

[0095] The MIC of bovine lactoferrin recombinant protein against Gram-negative bacteria was determined.

[0096] (1) Take a 96-well cell culture plate and add 100 μL of the corresponding broth to each well (96 wells in total) using a 100 μL pipette. Add 100 μL of bovine lactoferrin recombinant protein solution with initial concentrations of 2400 μg / mL, 2000 μg / mL, and 1600 μg / mL to the first well of each column in rows 1-8 (A1, B1, C1, D1, E1, F1, G1, H1), respectively.

[0097] (2) Using a pipette, draw 100 μL of liquid from the first well (A1-H1) of each row (AH) and transfer it to the second well. Mix thoroughly, then draw 100 μL from the second well and transfer it to the third well, and so on, serially diluting up to the tenth well. Discard 100 μL of liquid from the tenth well. Do not add protein solution to column 11 (positive control, containing only bacterial culture) and column 12 (negative control, without sterile culture or recombinant protein solution).

[0098] (3) Add 10 μL of pre-prepared 1×10^6 CFU / mL bacterial solution to each well in columns 1 to 11, mix gently, so that the final bacterial solution concentration in each well is about 1×10^5 CFU / mL, and do not add bacterial solution to column 12.

[0099] (4) Seal the 96-well plate with sealing film and incubate it in air at 37°C for 18±2 hours. Results can only be read when sufficient bacterial growth is shown in the positive control well (column 11). Compare the bacterial growth in each well with that in the positive control well to determine the lowest drug concentration that can significantly inhibit bacterial growth, i.e., the minimum inhibitory concentration (MIC). The results are shown in Table 2.

[0100] Table 2. MICs of bovine lactoferrin recombinant protein against different bacteria.

[0101]

[0102] As can be seen from the results in Table 2, bovine lactoferrin recombinant protein can effectively inhibit Escherichia coli and Salmonella.

[0103] Example 6

[0104] Evaluation of the antibacterial efficacy of a compound formulation containing streptococcal lysin and bovine lactoferrin.

[0105] (1) Staphylococcus aureus, Escherichia coli, Salmonella and Streptococcus suis were streaked onto TSA or NA medium plates and incubated at 37°C for 24 hours.

[0106] (2) Pick a single colony from each plate, inoculate it into TSB liquid medium, incubate at 37°C for 24 hours, measure the OD600 value of the bacterial solution, and dilute it with sterile medium to 1×10^6 CFU / mL for later use.

[0107] (3) Prepare a compound preparation of streptococcal lysin and bovine lactoferrin according to the formula shown in Table 3, and store it at 4 degrees Celsius.

[0108] (4) Take a 96-well cell culture plate and add 100 μL of liquid culture medium to each well using a 100 μL pipette. Add 100 μL of different concentrations of the compound preparation to the wells (A1-H1) in the first column of each row (AH). Take 100 μL of the solution from the first column of each row and transfer it to the second column. Mix well and then take 100 μL from the second column and transfer it to the third column. Continue this serial dilution up to the tenth column. Take 100 μL from the tenth column and discard it. The eleventh column is set as a positive control (containing only bacterial culture, no protein solution), and the twelfth column is set as a negative control (sterile culture, no protein solution).

[0109] (5) Add 10 μL of pre-prepared 1×10^6 CFU / mL bacterial solution to each well in columns 1 to 11, mix gently, so that the final bacterial solution concentration in each well is about 1×10^5 CFU / mL. No bacterial solution is added to column 12 as a negative control.

[0110] (6) Seal the 96-well plate with sealing film and incubate it in an air environment at 37°C for 18±2 hours. After incubation, check whether the positive control well (column 11) shows sufficient bacterial growth to ensure the validity of the experiment. Then compare the bacterial growth of each well with that of the positive control well and record the lowest concentration of compound protein that can significantly inhibit bacterial growth.

[0111] Table 3 Evaluation of the antibacterial effects of streptococcal lysin and bovine lactoferrin recombinant protein

[0112]

[0113] As can be seen from the results in Table 3, Formula 1 (0 μg / mL lysin, 2000 μg / mL bovine lactoferrin) had a MIC of 2000 μg / mL against Staphylococcus aureus and Escherichia coli, and 2000 μg / mL against Streptococcus suis, indicating that the antibacterial effect of bovine lactoferrin alone was poor (high MIC value).

[0114] Formula 2 (0 μg / mL lysin, 1600 μg / mL bovine lactoferrin): The MIC against Staphylococcus aureus, Escherichia coli, and Salmonella remained at 1600 μg / mL or was ineffective (MIC > 1600 μg / mL), indicating that the antibacterial effect was further weakened after reducing the concentration of bovine lactoferrin.

[0115] Formula 3 (512 μg / mL lyase, 0 μg / mL bovine lactoferrin): The MIC for Staphylococcus aureus was 32 μg / mL, for Salmonella it was 64 μg / mL, and it was ineffective against Escherichia coli and Streptococcus suis (MIC > 512 μg / mL), indicating that the lyase alone has limited effect against Gram-negative bacteria.

[0116] Formula 4 (512 μg / mL lysin, 1600 μg / mL bovine lactoferrin): MIC values ​​were 16 μg / mL for Staphylococcus aureus, 400 μg / mL for Escherichia coli, 400 μg / mL for Salmonella, and 32 μg / mL for Streptococcus suis. Compared to Formula 5, the MIC values ​​for Escherichia coli and Salmonella were higher (400 vs 250 μg / mL), indicating a slightly weaker antibacterial effect.

[0117] Formula 6 (256 μg / mL lyase, 2000 μg / mL bovine lactoferrin): The MIC values ​​were the same as those of Formula 5 (16 μg / mL for Staphylococcus aureus, 32 μg / mL for Streptococcus suis), but the MICs against Escherichia coli and Salmonella were as high as 2000 μg / mL, indicating that the antibacterial effect against Gram-negative bacteria was significantly reduced after lowering the concentration of lyase.

[0118] Formula 7 (64 μg / mL lyase, 2000 μg / mL bovine lactoferrin): The MIC value is similar to that of Formula 6, with a MIC of 2000 μg / mL against Escherichia coli and Salmonella, indicating poor antibacterial effect.

[0119] Formula 5 alone significantly enhanced the antibacterial effect against Gram-negative bacilli, while other formulas showed no significant improvement in antibacterial effect.

[0120] Example 7

[0121] Evaluation of the antibacterial efficiency of compound preparations

[0122] (1) Escherichia coli ATCC25923 and Staphylococcus aureus ATCC29213 were streaked onto TSA medium plates and incubated at 37°C for 24 hours.

[0123] (2) Pick a single colony and inoculate it into LB or TSB liquid medium. Incubate at 37°C with shaking (150 rpm) for 24 hours. Measure the OD600 value of the bacterial solution and dilute it with sterile medium to 1×10^6 CFU / mL for later use.

[0124] (3) Preparation of compound preparation: Streptococcal lysin (512 μg / mL) and bovine lactoferrin recombinant protein (2000 μg / mL) are mixed with sterile PBS or distilled water.

[0125] Prepare control group solutions: single lyase (512 μg / mL) and single bovine lactoferrin (2000 μg / mL).

[0126] All solutions should be prepared and used immediately, and stored at 4°C for short periods.

[0127] (4) Take a 96-well cell culture plate, add 100 μL of TSB liquid medium to each well, and set up the experiment according to the following groups (each group should be replicated at least 3 times):

[0128] Blank control group: Contains only bacterial suspension and culture medium, without antibacterial agents.

[0129] Compound formulation group: Add 10 μL of compound formulation (final concentration: lyase 51.2 μg / mL, bovine lactoferrin 200 μg / mL).

[0130] Single lyase group: Add 10 μL of lyase solution (final concentration 51.2 μg / mL).

[0131] Single bovine lactoferrin group: Add 10 μL of bovine lactoferrin solution (final concentration 200 μg / mL).

[0132] Add 10 μL of bacterial solution (1×10^6 CFU / mL) to each well, mix well, and make the final bacterial solution concentration approximately 1×10^5 CFU / mL.

[0133] At the same time, a negative control was set up: containing only culture medium, sterile solution and preparation.

[0134] (5) Seal the 96-well plate with sealing film and incubate it in a shaking incubator at 37°C (150 rpm). Take samples at 2, 4, 8, 12 and 24 hours respectively, and use a spectrophotometer to measure the OD600 value of each well. Record the bacterial growth. The results are shown in Table 4.

[0135] Table 4 Evaluation of antibacterial results using liquid co-culture method

[0136]

[0137] The results in Table 4 show that for Escherichia coli:

[0138] Single lysin: It had no significant inhibitory effect on Escherichia coli ATCC25923. The OD600 value continued to increase over time (reaching 3.589 after 24 hours), which was not significantly different from the blank control group (OD600=3.501 after 24 hours).

[0139] Single bovine lactoferrin: It showed some antibacterial activity, and completely inhibited bacterial growth after 12 hours of co-culture (OD600=0), but the OD600 value was still high in the early stage (2-8 hours) (e.g., OD600=0.014 at 8 hours), indicating that the antibacterial speed was slow.

[0140] The compound preparation showed a significant synergistic antibacterial effect, reducing the OD600 value to 0.054 in 2 hours and completely killing bacteria (OD600=0) in 8 hours, which is 4 hours faster than bovine lactoferrin alone.

[0141] For Staphylococcus aureus ATCC29213:

[0142] Single lyase: It has a certain antibacterial effect and can completely kill bacteria in 12 hours (OD600=0), but the early antibacterial effect is limited (OD600=0.061 after 4 hours).

[0143] Single bovine lactoferrin: It showed weak antibacterial activity, with an OD600 value of 0.799 after 24 hours, failing to completely kill bacteria, indicating that its inhibitory effect on Staphylococcus aureus is limited.

[0144] The compound formulation showed significantly better antibacterial effect than the single formulation. The OD600 value dropped to 0.025 in 2 hours and completely killed bacteria (OD600=0) in 4 hours, which was 8 hours faster than the single lysin, showing a very significant synergistic effect.

[0145] The results show that the combined formulation of streptococcal lysin and bovine lactoferrin exhibits significantly better antibacterial activity against *Escherichia coli* ATCC25923 and *Staphylococcus aureus* ATCC29213 than either the single lysin or bovine lactoferrin alone. The combined formulation showed a significant antibacterial effect within 2 hours and completely killed *E. coli* and *S. aureus* within 8 and 4 hours, respectively, demonstrating a much faster inhibition rate than the single formulations. The single lysin showed no antibacterial effect against *E. coli* and required 12 hours to completely kill *S. aureus*; bovine lactoferrin alone required 12 hours to completely kill *E. coli* and had a weaker antibacterial effect against *S. aureus*. The superior performance of the combined formulation may stem from the synergistic effect of the lysin disrupting the cell wall and bovine lactoferrin interfering with bacterial metabolism, providing experimental evidence for the development of highly effective antibacterial agents.

[0146] Example 8

[0147] 1. Screening for drug-resistant Escherichia coli (disk method)

[0148] (1) Streak the Escherichia coli isolate onto MH agar plates and incubate at 37°C for 24 hours. Pick a single colony and inoculate it onto LB or MH liquid medium and incubate at 37°C with shaking (150 rpm) for 24 hours. Measure the OD600 value of the bacterial culture and dilute it with sterile medium to 1×10^8 CFU / mL (approximately 0.5 McFarland standard).

[0149] (2) Prepare sterile filter paper discs (6 mm in diameter) and add the following antibiotic standard solutions (each disc contains the standard dose): ciprofloxacin (CIP, 5 μg), doxycycline (DX, 30 μg), gentamicin (GM, 10 μg), enrofloxacin (ENR, 5 μg), spectinomycin (SPT, 100 μg), and cefixime (CFM, 5 μg).

[0150] (3) Take 100 μL of bacterial suspension (1×10^8 CFU / mL), spread it evenly on the surface of MH agar plate, spread it evenly with a sterile spreader and let it dry. Use sterile tweezers to attach antibiotic paper discs to the surface of the plate (3 replicates for each antibiotic), and ensure that the distance between the paper discs is ≥24 mm.

[0151] (4) Invert the plate and incubate at 37°C for 24 hours. Measure the diameter of the inhibition zone (mm).

[0152] The screening results are shown in Table 5 and Figure 8 As shown.

[0153] Table 5 Results of in vitro antibacterial experiments on Escherichia coli isolates

[0154]

[0155] As can be seen from Table 5, this strain is only hyposensitive to cefixime and completely resistant to other antibiotics.

[0156] (5) Pick a single colony from the drug-resistant Escherichia coli plate, inoculate it into LB or MH liquid medium, and culture at 37°C with shaking (150 rpm) for 24 hours. Measure the OD600 value of the bacterial solution and dilute it with sterile medium to 1×10^8 CFU / mL for later use.

[0157] (6) Preparation of compound preparation: Streptococcal lysin (512 μg / mL) and bovine lactoferrin recombinant protein (2000 μg / mL) are mixed with sterile PBS or distilled water.

[0158] Prepare control group solutions: single lyase (512 μg / mL) and single bovine lactoferrin (2000 μg / mL).

[0159] (7) Prepare MH agar plates in advance, dry them overnight in an incubator at 37°C, take 100 μL of drug-resistant Escherichia coli bacterial solution (1×10^8 CFU / mL), spread it evenly on the surface of MH agar plates, spread it evenly with a sterile spreader, and then let it air dry.

[0160] (8) Use a pipette to take 2 μL of the compound preparation, single lyase, and single bovine lactoferrin and spot them on the surface of the plate (3 replicates for each preparation, with a spot spacing of ≥24 mm). Place the plate upright and incubate for 24 hours after the sample has been completely absorbed.

[0161] (9) Observe the formation of the inhibition zone on the plate and measure the diameter of the inhibition zone (mm, accurate to 0.1mm) with a vernier caliper.

[0162] The results are shown in Table 6 and Figure 9 As shown,

[0163] Table 6. Evaluation of the in vitro antibacterial efficacy of the compound formulation with bovine lactoferrin and lyase against drug-resistant Escherichia coli strains.

[0164]

[0165] Neither bovine lactoferrin (2000 μg / mL) nor streptococcal lysin (512 μg / mL) showed any inhibition zone (0 mm in diameter), indicating that they had no in vitro antibacterial effect against drug-resistant Escherichia coli.

[0166] The compound preparation (streptococcal lysin 512 μg / mL + bovine lactoferrin recombinant protein 2000 μg / mL) produced an inhibition zone of about 8 mm against drug-resistant Escherichia coli, indicating that it has certain in vitro antibacterial activity.

[0167] Compared to cefixime (in Table 5, the inhibition zone diameter is 10 mm, indicating low sensitivity), the combined formulation showed slightly weaker antibacterial effect, but it still effectively inhibited the growth of drug-resistant bacteria, demonstrating potential antibacterial application value. Further efforts could be made to enhance the antibacterial effect of the combined formulation by increasing its concentration, thereby enabling the development of novel anti-drug-resistant bacterial agents.

Claims

1. A compound antibacterial agent containing bovine lactoferrin and streptococcal lysin, characterized in that, The compound antibacterial preparation is composed of bovine lactoferrin and streptococcal lysin, wherein the concentration of bovine lactoferrin is 2000 μg / mL and the concentration of streptococcal lysin is 512 μg / mL. The bovine lactoferrin is a protein obtained by cloning the bovine lactoferrin gene sequence SEQ ID NO.2 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system. The streptococcal lysin is a protein obtained by cloning the streptococcal lysin gene sequence SEQ ID NO.1 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system.

2. The compound antibacterial preparation according to claim 1, characterized in that, The antibacterial spectrum of the compound antibacterial preparation includes Staphylococcus aureus, Staphylococcus discoloration, Staphylococcus epidermidis, cecal cocci, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, Escherichia coli, and Salmonella.

3. The antibacterial agent according to claim 2, characterized in that, The MIC of the antibacterial agent is 16 μg / mL against Staphylococcus aureus, 250 μg / mL against Escherichia coli, 250 μg / mL against Salmonella, and 32 μg / mL against Streptococcus suis.

4. The application of a composition containing bovine lactoferrin and streptococcal lysin in the preparation of an antibacterial agent, characterized in that, In the composition, the concentration of bovine lactoferrin is 2000 μg / mL, and the concentration of streptococcal lysin is 512 μg / mL. The bovine lactoferrin is a protein obtained by cloning the bovine lactoferrin gene sequence SEQ ID NO.2 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system. The streptococcal lysin is a protein obtained by cloning the streptococcal lysin gene sequence SEQ ID NO.1 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system. The antibacterial spectrum of the antibacterial agent includes Staphylococcus aureus, Staphylococcus discoloration, Staphylococcus epidermidis, cecal cocci, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus lactis, Escherichia coli, and Salmonella.

5. The use of a composition of bovine lactoferrin and streptococcal lysin in the preparation of a medicament for inhibiting drug-resistant Escherichia coli, characterized in that, In the composition, the concentration of bovine lactoferrin is 2000 μg / mL, and the concentration of streptococcal lysin is 512 μg / mL. The bovine lactoferrin is a protein obtained by cloning the bovine lactoferrin gene sequence SEQ ID NO.2 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system. The streptococcal lysin is a protein obtained by cloning the streptococcal lysin gene sequence SEQ ID NO.1 into the pFastBac1 vector and then expressing it through a baculovirus-insect cell eukaryotic expression system. The drug-resistant Escherichia coli is an isolate of Escherichia coli that is resistant to ciprofloxacin, doxycycline, gentamicin, enrofloxacin, and spectinomycin, but only has low sensitivity to cefixime.

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