A recombinant strain of high-yield duck riiberger bacillus phage lytic enzyme, a mutant strain, a construction method and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a recombinant strain of high-yield duck Riemerella anatipestifer phage lytic enzyme, a mutant strain, a construction method and application. The application provides a coding gene of duck Riemerella anatipestifer phage lytic enzyme, and further constructs a recombinant Lactococcus lactis of high-yield duck Riemerella anatipestifer phage lytic enzyme. The mutant strain provided by the application is obtained by using the recombinant Lactococcus lactis as a starting strain, through mutagenesis breeding technology and specific high-throughput screening strategies, has higher secretion expression of duck Riemerella anatipestifer phage lytic enzyme, and has good genetic stability in an antibiotic-free culture medium, and is suitable for industrialized and efficient production of active lytic enzyme. The duck Riemerella anatipestifer phage lytic enzyme expressed by the mutant strain has obvious lytic effect on duck Riemerella anatipestifer in vitro, has significant prevention and treatment effect on duck Riemerella anatipestifer infection in vivo, can effectively improve the survival rate of infected chicks, and can remove pathogenic bacteria in the body.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant strain, mutant strain, construction method, and application of a high-yield duck plague Riesella phage lysin. Background Technology

[0002] Duck infectious serositis (also known as duck disease caused by Riedelella anatipestifer) is caused by Riedelella anatipestifer (… Riemerella anatipestifer Bacterial phage lysinic pneumonia (BPH) is a contagious, acute, or chronic septicemic disease caused by bacteria, and is one of the most serious bacterial diseases affecting poultry farming, including ducks, geese, and turkeys, worldwide. Characterized by high morbidity and mortality rates in ducklings, it causes significant economic losses to the poultry industry. For a long time, antibiotics have been the primary means of controlling this disease; however, the widespread and even overuse of antibiotics has led to the frequent emergence of multidrug-resistant strains, making clinical treatment increasingly difficult. At the same time, antibiotic residues pose a potential threat to food safety and public health. Phage lysins, as a novel antibacterial agent, demonstrate great potential as an alternative to antibiotics due to their high efficiency, strong specificity, and low tendency to induce resistance.

[0003] Producing *Riegeria anatipestifer* phage lysins using genetically engineered bacteria is crucial for their commercial application. *Escherichia coli* is currently the most commonly used and mature prokaryotic protein expression system, and the preferred platform for producing various phage lysins. Existing literature reports the successful prokaryotic expression of the *Riegeria anatipestifer* phage lysin gene in *E. coli* strain BL21(DE3). However, as a Gram-negative bacterium, *E. coli* contains a large amount of lipopolysaccharide (LPS), or endotoxin, in its outer membrane. Endotoxin is a potent pyrogen and immunostimulant; even trace amounts present in the final veterinary drug product can cause serious adverse reactions in animals, such as fever and shock. Therefore, a dedicated step must be included in the downstream purification process to remove endotoxin, increasing the complexity of the production process, extending the production cycle, and significantly increasing production costs. Furthermore, to achieve high yields, genetically engineered bacteria typically require high-density fermentation, but high-density cell density produces large amounts of metabolic byproducts (such as acetic acid), inhibiting cell growth and protein expression. Summary of the Invention

[0004] To address the above problems, this invention provides a recombinant strain, mutant strain, construction method, and applications of high-yield *R. anatipestifer* phage lysin. The recombinant strain provided by this invention is constructed using *Lactococcus lactis*, a food-grade safe microorganism, as the starting strain. It can secrete and express active *R. anatipestifer* phage lysin, is safe and antibiotic-free, and can stably colonize in the animal intestine. While expressing the *R. anatipestifer* phage lysin to exert its bactericidal effect, it can also help improve the intestinal health of animals. The mutant strain provided by this invention has a higher expression level of *R. anatipestifer* phage lysin and good genetic stability, showing good prospects for industrial application.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a gene encoding a bacteriophage lysin of *Riebrio anatidae*, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0007] Among microbial expression hosts, *Lactococcus lactis* is a recognized food-grade safe (GRAS) strain, non-pathogenic and capable of stably colonizing the animal gut. While expressing exogenous genes, it can act as a probiotic to regulate the intestinal microecology of animals and help improve the intestinal health of farmed animals. Currently, a mature system has not yet been established for technologies related to expressing *Riegeria anatipestifer* phage lysins using *Lactococcus lactis* as a host.

[0008] This encoding gene was artificially designed based on the codon bias of Lactococcus lactis, taking into account the gene sequence characteristics of the Rieslerella anatipestifer phage lysin. It can achieve high expression levels and activity in Lactococcus lactis.

[0009] A second aspect of the present invention provides a recombinant lactococcus that secretes and expresses an anatipestifer phage lysin. This recombinant lactococcus is constructed from a *Lactococcus lactis* strain by transfecting it with an antibiotic-free recombinant expression plasmid containing the aforementioned coding gene. The recombinant expression plasmid carries a signal peptide SP. USP45 The fusion gene includes the coding sequence, the nucleotide sequence shown in SEQ ID No. 1, and the 6×His tag coding sequence.

[0010] Preferably, the nucleotide sequence of the fusion gene is shown in SEQ ID No. 2.

[0011] Preferably, the starting strain is *Lactococcus lactis* NZ3900. *Lactococcus lactis* NZ3900 has a clear genetic background and strong tolerance for foreign genes. It does not contain natural plasmids, thus avoiding homologous recombination with foreign vectors and providing an excellent host environment for stable expression of foreign genes. Furthermore, the nucleotide sequence shown in SEQ ID No. 1 conforms to the codon preference of *Lactococcus lactis* NZ3900.

[0012] Preferably, the starting plasmid of the recombinant expression plasmid is expression plasmid pNZ8149. Expression plasmid pNZ8149 is a commonly used inducible vector in Lactococcus lactis expression systems. It not only carries the nisA promoter, allowing for efficient induction and regulation via Nisin to achieve controllable expression of exogenous proteins, but also possesses lactose auxotroph selection markers and multiple cloning sites, facilitating the cloning of the *R. anatidae* phage lyase gene and the construction of antibiotic-free label-free expression vectors. Furthermore, it exhibits high replication stability in *Lactococcus lactis*, effectively ensuring that the target gene is not lost during the passage of the recombinant strain. Moreover, the nucleotide sequence shown in SEQ ID No. 1 is compatible with the expression regulatory mechanism of expression plasmid pNZ8149, thereby achieving high expression of the *R. anatidae* phage lyase.

[0013] A third aspect of this invention provides a method for constructing the above-mentioned recombinant lactococcus, comprising the following steps: S1. The fusion gene is cloned into the expression plasmid to construct the recombinant expression plasmid; S2. The recombinant expression plasmid is transferred into competent cells of the starting strain, and positive recombinant strains are obtained by lactose screening, which are the recombinant lactococci.

[0014] Preferably, the method for transferring the recombinant expression plasmid into competent cells of the starting strain is electroporation.

[0015] The fourth aspect of this invention provides a high-yield *Lactococcus lactis* mutant strain SJ-LI-LYS-001 producing *Riebelella anatipestifer* phage lysin, its taxonomic name being *Lactococcus lactis* (…). Lactococcus lactis It was deposited on January 9, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37338.

[0016] The aforementioned recombinant *Lactococcus lactis* can achieve the secretory expression of *Riebelella anatipestifer* phage lysin. If the expression level can be further increased, it can better meet the cost and efficiency requirements of industrial production. Mutagenesis breeding is a classic industrial microbial improvement technique, but in the field of *Lactococcus lactis* protein expression, especially for food-grade engineered bacteria without antibiotic resistance markers, it lacks efficient and precise screening methods. Traditional methods cannot rapidly screen out a very small number of high-yielding strains of exogenous proteins from a large number of mutants without the use of antibiotics.

[0017] The mutant strain SJ-LI-LYS-001 was obtained by using the above-mentioned recombinant lactococcus lactis as the starting strain, performing ultraviolet mutagenesis, and then screening through primary screening in hyperosmolar medium and secondary screening after induced expression. Compared with the starting strain, the expression level of phage lysin of Rieslinger anatipestifer was significantly increased, and the genetic stability was good, making it more promising for industrial application.

[0018] The fifth aspect of this invention provides the use of the above-mentioned recombinant Lactococcus lactis or Lactococcus lactis mutant strain SJ-LI-LYS-001 in the prevention and / or treatment of Riegeria duckweed infection in avian animals.

[0019] The aforementioned *Lactococcus lactis* mutant strain SJ-LI-LYS-001 can effectively eliminate *R. anatipestifer* in target organs (such as the liver and brain) of infected poultry, effectively playing a preventive and therapeutic role, significantly reducing mortality and increasing survival rates in infected poultry. The aforementioned recombinant *Lactococcus lactis* can also express *R. anatipestifer* phage lysin, thus also having a preventive and therapeutic effect against *R. anatipestifer* infection in poultry.

[0020] Preferably, the poultry is a young bird.

[0021] More preferably, the poultry includes chicks.

[0022] The sixth aspect of this invention provides a method for expressing *R. anatipestifer* phage lysin using the above-mentioned recombinant *Lactococcus lactis* or the *Lactococcus lactis* mutant strain SJ-LI-LYS-001, comprising the following steps: fermenting the recombinant *Lactococcus lactis* or the *Lactococcus lactis* mutant strain SJ-LI-LYS-001, followed by induction culture using Nisin. The resulting induction culture medium contains the above-mentioned *R. anatipestifer* phage lysin.

[0023] Optionally, the fermentation culture method is as follows: the seed culture of the recombinant Lactococcus lactis or the Lactococcus lactis mutant strain SJ-LI-LYS-001 is inoculated into the fermentation medium, and fermented for 6 hours under positive pressure, temperature of 30℃, pH of 6.95~7.0, and rotation speed of 50 r / min. The fermentation medium consists of: lactose 10 g / L, peptone 15 g / L, yeast extract 20 g / L, beef extract 20 g / L, calcium chloride 0.01 g / L, magnesium sulfate heptahydrate 0.2 g / L, and MnSO4·H2O 0.05 g / L.

[0024] Preferably, during the induction culture, the amount of Nisin added is 10~20 ng / mL.

[0025] Preferably, during the induction culture, a 30% lactose aqueous solution is added at a rate of 30 mL / L / h to achieve efficient production of Riesler's duck phage lysin.

[0026] The beneficial effects of this invention are: 1. This invention successfully constructed a food-grade engineered bacterium capable of secreting and expressing an active *R. anatidae* phage lysin, which is safe and antibiotic-free. While expressing the *R. anatidae* phage lysin to exert its bactericidal effect, this engineered bacterium can also help improve the intestinal health of animals, achieving a dual effect of "disease control + intestinal conditioning".

[0027] 2. This invention successfully obtained the mutant strain SJ-LI-LYS-001, which exhibits significantly increased secretion and expression of *R. anatipestifer* phage lysin, through efficient mutagenesis breeding and a combination of hyperosmolar primary screening and immunoblotting secondary screening. The *R. anatipestifer* phage lysin produced by this mutant strain and its originating strain not only demonstrates significant lytic activity against *R. anatipestifer* in vitro, exhibiting high lytic activity, but also shows excellent preventive and therapeutic effects in animals, and possesses good genetic stability. It can be used to develop safe and efficient biocontrol products for *R. anatipestifer* disease and has promising application prospects. Attached Figure Description

[0028] Figure 1 This is the Western blot verification result in Example 2 of the present invention; where lane 1 is the Maker and lane 2 is the supernatant after induction with 10 ng / mL (final concentration) Nisin; Figure 2 This invention demonstrates the in vitro antibacterial effect of empty vector bacteria and recombinant Lactococcus lactis in Example 4. Well 1 contained physiological saline, well 2 contained the induction culture supernatant of the empty vector bacteria, and well 3 contained recombinant Lactococcus lactis pNZ8149-SP. USP45 -lys induction culture supernatant; Figure 3The strain SJ-LI-LYS-001 and recombinant lactococcus pNZ8149-SP in Example 5 of this invention. USP45 Western blot grayscale analysis results of the induction culture medium of strain -lys; lane 1 is Maker, and lane 2 is recombinant lactococcus pNZ8149-SP. USP45 -lys induction culture supernatant, lane 3 is the induction culture medium of mutant strain SJ-LI-LYS-001; Figure 4 The mutant strain SJ-LI-LYS-001 and recombinant lactococcus pNZ8149-SP in Example 6 of this invention are examples of this invention. USP45 Comparison of the antibacterial effects of -lys and empty vector bacterial induction culture supernatant on *Riebelella anatipestifer*; well 1 contains the induction culture supernatant of empty vector bacteria, and well 2 contains recombinant *Lactococcus lactis* pNZ8149-SP. USP45 The induction culture supernatant of -lys was added to well 3, and the induction culture supernatant of mutant strain SJ-LI-LYS-001 was added to well 3.

[0029] The Lactococcus lactis mutant strain SJ-LI-LYS-001 is classified and named Lactococcus lactis ( Lactococcus lactis (), deposited by: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 37338, deposit date: January 9, 2026. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0031] duck plague bacillus ( Riemerella anatipestifer Bacterial phage is a major pathogen causing huge economic losses to the global waterfowl industry, and its increasingly serious antibiotic resistance problem poses a severe challenge to traditional control strategies. Phage lysins, as a novel antibacterial agent, show great potential to replace antibiotics due to their advantages such as high efficiency, high specificity, and low resistance to induction. *Escherichia coli* is the most commonly used recombinant expression chassis cell, but its application faces challenges such as endotoxin risks and high purification costs. Furthermore, the high-density fermentation commonly used in production can inhibit the growth and protein expression of genetically engineered bacteria.

[0032] This invention provides a gene encoding a phage lyase from *Riesella dysenteriae*, the nucleotide sequence of which is shown in SEQ ID NO. 1. This gene was artificially designed based on the codon bias of *Lactococcus lactis*, a food-grade safe microorganism, targeting the gene sequence characteristics of *Riesella dysenteriae* phage lyase, and can achieve high expression levels and activity in *Lactococcus lactis*.

[0033] This invention also provides a recombinant lactococcus that secretes and expresses anaerobes bacteriophage lysin and a method for constructing it.

[0034] This invention also provides a high-yield lactococcal mutant strain SJ-LI-LYS-001 that produces duck plague Riesella phage lysin.

[0035] This invention also provides the application of the above-mentioned recombinant Lactococcus lactis or Lactococcus lactis mutant strain SJ-LI-LYS-001 in the prevention and / or treatment of Riegeria duckweed infection in avian animals.

[0036] This invention also provides a method for expressing Rieslerella anatipestifer phage lysin using the above-mentioned recombinant Lactococcus lactis or Lactococcus lactis mutant strain SJ-LI-LYS-001.

[0037] The present invention will be described below through specific embodiments.

[0038] The culture medium components used in the following examples are: M17 broth medium (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.): soybean peptone 5.0 g / L, peptone 2.5 g / L, casein peptone 2.5 g / L, yeast extract 2.5 g / L, beef extract 5.0 g / L, lactose 5.0 g / L, sodium ascorbate 0.5 g / L, sodium β-glycerophosphate 19.0 g / L, magnesium sulfate 0.25 g / L, pH 7.2 ± 0.2 (25℃); M17 agar medium (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.): soybean peptone 5.0 g / L, peptone 2.5 g / L, casein peptone 2.5 g / L, yeast extract 2.5 g / L, beef extract 5.0 g / L, lactose 5.0 g / L, sodium ascorbate 0.5 g / L, sodium β-glycerophosphate 19.0 g / L, magnesium sulfate 0.25 g / L, agar 12.75 g / L, pH 7.2 ± 0.2 (25℃); Fermentation medium: lactose 10g / L, peptone 15g / L, yeast powder 20g / L, beef powder 20g / L, calcium chloride 0.01g / L, magnesium sulfate heptahydrate 0.2g / L, MnSO4·H2O 0.05g / L; M17 glucose medium: soybean peptone 5.0 g / L, peptone 2.5 g / L, casein peptone 2.5 g / L, yeast extract 2.5 g / L, beef extract 5.0 g / L, glucose 5.0 g / L, sodium ascorbate 0.5 g / L, sodium β-glycerophosphate 19.0 g / L, magnesium sulfate 0.25 g / L, pH 7.2 ± 0.2 (25℃).

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Example 1 This embodiment provides a recombinant expression plasmid containing the gene encoding the phage lyase of *Riesella dysporidis* as shown in SEQ ID No. 1, as well as a recombinant lactococcus that secretes and expresses the phage lyase of *Riesella dysporidis* and its construction method.

[0041] 1. Extract the expression plasmid pNZ8149 using a plasmid extraction kit, and digest the plasmid with NocI and KpnI respectively.

[0042] 2. CC (which forms a NocI restriction site with ATGG at the beginning of the sequence of SEQ ID No. 2) and KpnI restriction site were added to the beginning and end of the fusion gene as shown in SEQ ID No. 2, respectively. Then, 15 homologous arms of plasmid pNZ8149 were added to the beginning and end to obtain the sequence shown in SEQ ID No. 3, which was sent to Shanghai Sangon Biotech Co., Ltd. for synthesis.

[0043] 3. The gene fragment with the sequence shown in SEQ ID No. 3 and the expression vector pNZ8149 were ligated to obtain the recombinant expression plasmid.

[0044] 4. Electroporate the recombinant expression plasmid obtained in step 3 into Lactococcus lactis NZ3900, screen for positive transformants, and obtain recombinant Lactococcus lactis expressing Riesler's anatipestifer phage lysin (named pNZ8149-SP). USP45 -lys).

[0045] Example 2 This embodiment provides the recombinant lactococcus pNZ8149-SP strain constructed using Example 1. USP45 -lys method for expressing Riegeria anatipestifer phage lyase.

[0046] Fresh recombinant lactococcus pNZ8149-SP USP45-lys bacterial culture was inoculated at a ratio of 2% into M17 broth medium and incubated statically at 30℃ for 16 h; then, it was inoculated a second time at a ratio of 5% into M17 broth medium and cultured until the recombinant lactococcus reached the logarithmic growth phase. Nisin was added to a final concentration of 10 ng / mL, and the culture was incubated statically at 30℃ for 10 h for induction. After induction, the supernatant was collected for Western blot analysis.

[0047] The procedure for Western blot detection is as follows: Immerse a standard SDS-PAGE gel plate in electrophoresis transfer buffer for 15 minutes; wearing gloves, cut filter paper and electrophoresis membrane (to prevent contamination), and immerse them in electrophoresis transfer buffer for 10 minutes to remove air bubbles. Place the transfer cassette in a shallow dish, place a sponge pad soaked in electrophoresis transfer buffer on the cassette wall, and place a moistened filter paper on top; carefully place the gel (to prevent air bubbles), add another soaked sponge, close the cassette and place it in the transfer tank. Place the transfer tank in an ice box, fill it with 4°C pre-cooled electrophoresis transfer buffer, and transfer at a constant current of 200mA for 1.5 hours. Peel off the transfer membrane and mark it (e.g., cut a notch in the upper left corner), rinse three times with TBS-T at room temperature (5 minutes each time) to remove SDS. Prepare blocking solution by adding 5g of blocking agent to 100ml of double-distilled water, and block the membrane in an internal shaker at room temperature for 30 minutes; rinse three times with pH 7.6 TBS-T. Place the membrane in a hybridization bag, add primary antibody solution, seal, and incubate overnight at 4°C. Rinse three times with TBS-T at pH 7.6; add 2 μl of HRP-labeled secondary antibody to 10 ml of diluent to prepare working solution, place the membrane in a bag, add the solution and seal, incubate at 37°C for 1 hour. Rinse three times with TBS-T at pH 7.6. Add 200 μl each of DAB chromogenic solutions A and B to 4 ml of TBS-T, mix well, add to the front side of the membrane, and develop at room temperature. Stop the process when the target band appears and there is no background. Results are as follows. Figure 1 As shown, a band appears at molecular weight 21KD, indicating that the supernatant of the recombinant lactococcus obtained in Example 1 contains Rieslingerella anatipestifer phage lysin.

[0048] Example 3 This embodiment provides the recombinant lactococcus pNZ8149-SP strain constructed using Example 1. USP45 -lys method for high-density fermentation-induced expression of Riesler's duck phage lyase.

[0049] Recombinant Lactococcus lactis pNZ8149-SP USP45-lys was inoculated at a 1% ratio into 15 mL test tubes containing seed culture medium (M17 broth), and incubated statically at 30°C for 19 h. Then, it was inoculated at a 5.0% ratio into shake flasks containing seed culture medium and incubated for 8 h to obtain the seed culture. The seed culture was then inoculated into fermentation medium at a 10% inoculation rate. Positive pressure was maintained at 30°C. After inoculation, the pH was adjusted to 7.0, and ammonia was continuously added to maintain the pH between 6.95 and 7.0. The fermentation speed was 50 rpm. After 6 h of fermentation, 10 ng / mL Nisin was added for induction culture, and simultaneously, 30% lactose solution was added at 30 mL / L / h. Induction culture was completed after 11 h, and the viable cell count in the induction culture reached 1.5 × 10⁻⁶. 10 / mL or higher. The induction culture medium was centrifuged at 5000 rpm and 4℃ for 15 min, and the content of duck plague Riedelella phage lysin in the obtained induction culture supernatant was 37.9 mg / L.

[0050] Example 4 This embodiment provides the recombinant lactococcus pNZ8149-SP strain constructed in Example 1. USP45 The in vitro antibacterial effect of the phage lyase of *Riebrio anatidae* expressed by -lys.

[0051] The *Riebelella anatipestifer* was activated and cultured to an OD600 of 0.3, and then spread 100 μL onto a TSA plate. After evenly punching wells in the TSA plate, 200 μL of recombinant *Lactococcus lactis* pNZ8149-SP was added to each well. USP45 The induction culture supernatant of -lys (prepared according to Example 3, filtered and sterilized before being added to the wells), with an equal volume of the induction culture supernatant of the empty vector bacteria without the target gene (prepared according to Example 3, filtered and sterilized before being added to the wells) and physiological saline as controls, was incubated at 37°C for 24 h to observe the antibacterial effect. The empty vector bacteria were constructed by electroporating the expression vector pNZ8149 into Lactococcus lactis NZ3900.

[0052] The results are as follows Figure 2 As shown.

[0053] Example 5 This embodiment provides the Lactococcus lactis mutant strain SJ-LI-LYS-001, which produces high levels of anatipestifer phage lysin, and the process by which it was obtained.

[0054] 1. UV mutagenesis treatment 1) The recombinant lactococcus pNZ8149-SP strain obtained in Example 1 was used. USP45 -lys were inoculated onto M17 agar medium, and after culturing, recombinant lactococcus pNZ8149-SP was picked from the M17 agar medium. USP45A single colony of -lys was inoculated into 5 mL of M17 broth and incubated at 30°C overnight.

[0055] 2) Transfer 1% inoculum to 50 mL of fresh M17 lactose medium and incubate at 30°C until mid-log (OD100). 600 ≈0.5).

[0056] 3) Take 20 mL of bacterial culture, centrifuge at 4 °C to collect the bacterial cells, wash twice with pre-cooled sterile physiological saline, and finally resuspend in 20 mL of physiological saline.

[0057] 4) Preheat a 15W UV lamp (254nm) for 20 minutes. Take 5mL of bacterial suspension in a sterile empty petri dish, place it 30cm under the UV lamp, turn on magnetic stirring, and irradiate for 30 seconds (this condition has been determined through preliminary experiments, and the lethality rate is about 80%).

[0058] 5) Immediately after irradiation, wrap the bacterial suspension with aluminum foil to protect it from light. Dilute the bacterial suspension appropriately, spread it on M17 lactose plates, and incubate at 30°C in the dark until a large number of single colonies are formed.

[0059] 2. High-throughput screening: 1) Initial screening (hyperosmolarity screening): Approximately 600 single colonies were randomly selected from the mutagenized plates and inoculated onto both standard M17 agar and M17 agar containing 6% sucrose using toothpicks. After incubation at 30°C for 48 hours, clones that grew significantly faster on the sucrose-containing M17 agar than the surrounding colonies and grew normally on the standard M17 agar were selected, yielding a total of 120 primary mutant strains, which were then inoculated into 96-well deep-well plates for further culture.

[0060] 2) Secondary screening (Western Blot quantitative screening): Primary mutant strains were microcultured in 96-well deep-well plates (M17 agar medium supplemented with 20 ng / mL Nisin). After induction, the culture medium was centrifuged at 5000 rpm and 4°C for 15 min, and the supernatant was collected. The OD600 of each well was measured for standardization. Supernatant samples with equal biomass were analyzed by SDS-PAGE and Western blotting, using anti-His-tagged antibody as the primary antibody. By comparing the band grayscale with a known concentration of purified *R. anatidae* phage lyase standard, eight *R. anatidae* strains were found to have higher phage lyase expression levels than recombinant *Lactococcus lactis* pNZ8149-SP. USP45 The candidate mutant strains of -lys were named SJ-LI-LYS-001, and the mutant with the highest expression level was named SJ-LI-LYS-001.

[0061] 3. Verification and genetic stability testing: SJ-LI-LYS-001 and recombinant lactococcus pNZ8149-SP USP45 -lys was subjected to parallel shake-flask fermentation (method as in Example 2). The supernatant from the induction culture was analyzed by Western blotting. The amount of *Riebelella anatipestifer* phage lyase protein secreted by strain SJ-LI-LYS-001 was approximately the same as that of recombinant *Lactococcus lactis* pNZ8149-SP. USP45 -lys is 1.9 times that of lys. For example... Figure 3 As shown.

[0062] SJ-LI-LYS-001 was passaged 15 times in M17 glucose medium, and its growth and lyase expression levels were assessed every 3 generations on M17 agar medium. The results showed that the recombinant expression plasmid obtained by ligating the gene fragment with the sequence shown in SEQ ID No. 3 and the expression vector pNZ8149 had a retention rate of over 98%, and the high-yield trait was stably inherited.

[0063] Example 6 This embodiment demonstrates the in vitro antibacterial effect of the mutant strain SJ-LI-LYS-001.

[0064] The mutant strain SJ-LI-LYS-001 and recombinant Lactococcus lactis pNZ8149-SP were used. USP45 -lys and the empty vector bacteria were subjected to parallel fermentation and induction cultures according to the method described in Example 3. Using the plate-punching method, an equal volume of induction culture supernatant was added to the wells of a TSA plate coated with *Riebelella anatipestifer* (OD600=0.3, 200 μL), with the empty vector bacteria induction culture supernatant serving as a negative control. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured. The empty vector bacteria were constructed by electroporating the expression vector pNZ8149 into *Lactococcus lactis* NZ3900.

[0065] The results are as follows Figure 4 As shown, the supernatant from the induction culture of the mutant strain SJ-LI-LYS-001 produced an inhibition zone with a diameter of 2.6 cm, which was significantly larger than that of the recombinant lactococcus pNZ8149-SP. USP45 -lys showed a 1.5cm inhibition zone, while the negative control group showed no inhibition zone.

[0066] Example 7 This example tested the mutant strain SJ-LI-LYS-001 and the recombinant Lactococcus lactis pNZ8149-SP. USP45 Protein content of Riegeria anatipestifer phage lyase in -lys induced culture medium.

[0067] 1. Concentration of induction culture supernatant and determination of total protein (1) The mutant strain SJ-LI-LYS-001 and recombinant lactococcus pNZ8149-SP were prepared according to the method in Example 3. USP45 The induction culture medium of -lys was centrifuged at 4℃ and 12,000 r / min for 15 min, and the supernatant was collected.

[0068] (2) The supernatant was concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa (to 1 / 20 of the original volume).

[0069] (3) The total protein concentration of the concentrate was determined using the Bradford method (Coomassie Brilliant Blue method). A standard curve was plotted using bovine serum albumin (BSA) as the standard (concentration range: 0-2000 μg / mL). An appropriate amount of the concentrate was diluted and mixed with Coomassie Brilliant Blue G-250 staining solution. The absorbance at 595 nm was measured, and the total protein concentration of the concentrate (unit: mg / mL) was calculated based on the standard curve.

[0070] 2. SDS-PAGE separation and target protein percentage analysis (1) Prepare 12% separating gel and 5% stacking gel.

[0071] (2) Sample loading: Take a concentrated sample equivalent to 20 μg of total protein, mix it with an equal volume of 2×SDS-PAGE loading buffer, heat in a boiling water bath for 5 min, and then load the sample. At the same time, load different concentrations of BSA standards (1, 2, 5 μg) to prepare grayscale standard curves and pre-stained protein molecular weight standards (Marker).

[0072] (3) Electrophoresis: Electrophoresis is performed at a constant voltage of 80V until the boundary between the stacking gel and the separating gel is reached. Then, the voltage is changed to 120V and electrophoresis is continued until the bromophenol blue reaches the bottom of the gel.

[0073] (4) Staining and destaining: After electrophoresis, the gel was placed in 0.1% Coomassie Brilliant Blue R-250 staining solution and stained on a shaker for 1 hour. Then it was transferred to destaining solution (acetic acid:methanol:water = 1:3:6) and destained on a shaker until the background was transparent and the bands were clear.

[0074] 3. Calculation of target protein content (1) Scan the decolorized gel using a gel imaging system.

[0075] (2) The electrophoresis pattern was analyzed using image analysis software (such as Image Lab). A clear target protein band was visible at the 21 kDa position. The gray values ​​of the target band and all protein bands in the same lane were measured.

[0076] (3) Calculate the percentage of the gray value of the target protein band to the total gray value of the protein band in the lane, i.e., the percentage of the target protein to the total protein (%).

[0077] (4) The concentration of the target protein in the induction culture medium is calculated according to the following formula: The concentration of lyase in the induction culture medium (mg / mL) = the total protein concentration of the concentrate (mg / mL) × the percentage of target protein (%) / the concentration factor.

[0078] 4. Measurement Results Recombinant Lactococcus lactis pNZ8149-SP USP45 The phage lyase content of *R. anatidae* in the supernatant induced by the *-lys* strain was approximately 37.9 mg / L, while that of the mutant strain SJ-LI-LYS-001 reached 76.3 mg / L, representing an expression level approximately twice that of the original strain. This result is consistent with the trend of increasing inhibition zone diameter in the supernatant induced by the mutant strain SJ-LI-LYS-001 in Example 6, demonstrating that the mutant strain SJ-LI-LYS-001, compared to its original strain, recombinant *Lactococcus lactis* pNZ8149-SP, exhibits a significant advantage. USP45 -lys significantly increased the expression level of phage lyase in Rieslerella anatipestifer.

[0079] Example 8 This embodiment investigated the preventive and therapeutic effects of the mutant strain SJ-LI-LYS-001 on diseases caused by Riegeria duckweed infection in a chick model.

[0080] 1. Experimental Materials and Methods 1.1 Laboratory Animals and Grouping Sixty healthy one-day-old chicks were randomly divided into four groups of 15 each: Control group: No infection was detected, and patients received oral sterile PBS.

[0081] Infection group: infected with Riedelella anatipestifer, and orally administered sterile PBS as a control.

[0082] Prevention group: Oral administration of recombinant duck plague Riesella phage lysin preparation before infection.

[0083] Treatment group: After infection, patients were given oral recombinant duck plague Riedelium phage lysin preparation.

[0084] 1.2 Strains and Riegeria anatipestifer phage lysin The target strain was *Riekelium anatipestifer*. Preliminary experiments determined its median lethal dose (LD50) for 1-day-old chicks to be 1.5 × 10⁻⁶. 8 CFU / mL.

[0085] Recombinant duck plague riezobacter phage lysin preparation: The induction culture supernatant of mutant strain SJ-LI-LYS-001 was prepared according to the method in Example 3, and sterilized by filtration through a 0.22 μm filter membrane. Its lysin activity (titer ≥10) was measured before use. 9 U / mL).

[0086] 1.3 Experimental Treatment The specific processing flow is shown in Table 1: Table 1. Experimental grouping and treatment schemes for chicks

[0087] Note: The oral dose of the lyase preparation is 0.1 mL / animal / day, administered once daily.

[0088] 1.4 Observation Indicators and Testing 1. Clinical symptoms and survival rate: Observe for 7 consecutive days, record the mental state, feed intake, mortality and other conditions of chicks in each group, and calculate the survival rate.

[0089] 2. Pathogen clearance effect (tissue bacterial load determination): At the end of the experiment, surviving chicks were euthanized, and liver and brain tissues were aseptically collected. After weighing, the tissues were homogenized with sterile PBS. The homogenate was serially diluted 10-fold, and 100 μL was spread on TSA plates containing 5% newborn calf serum. After incubation at 37°C for 18-24 h, colony-forming units (CFU) were counted, and the bacterial load per gram of tissue (CFU / g) was calculated.

[0090] 2. Results 2.1 Clinical symptoms and protection rate In the infected group: 2 hours after infection, lethargy and loss of appetite began to appear; subsequently, the individuals huddled together, became inactive, and had serous or mucous discharge from the eyes and nose; death occurred within 12 hours, accompanied by symptoms such as convulsions and opisthotonus before death, with the peak mortality occurring 2-3 days later. The final survival rate was only 40%.

[0091] Prevention and treatment groups: After infection, both groups experienced only brief periods of lethargy and loss of appetite, followed by a rapid return to normal activity and eating. The survival rate was 93.3% in the prevention group and 86.7% in the treatment group.

[0092] Control group: All members survived healthily, with a survival rate of 100%.

[0093] The results showed that both preventative and therapeutic administration of the supernatant of the mutant strain SJ-LI-LYS-001 significantly reduced chick mortality caused by Riegeria duckweed infection (P<0.01), with the preventative effect being slightly better than the therapeutic effect.

[0094] 2.2 Pathogen clearance effect Bacterial isolation results from the livers and brains of surviving chickens showed: In the infected group: the detection rate of Riekelella anatipestifer in the liver of surviving chickens was 100%, and the detection rate in the brain was 66.7%.

[0095] Prevention group vs. treatment group: The detection rates of Riegeria duckweed in the liver and brain of the prevention group were 14.3% and 7.1%, respectively; the detection rates of Riegeria duckweed in the liver and brain of the treatment group were 23.1% and 7.7%, respectively.

[0096] The results of tissue bacterial load assays showed that the bacterial load in the liver and brain of infected chicks was significantly higher than that in the prevention and treatment groups. P <0.01), the supernatant of the induction culture of the mutant strain SJ-LI-LYS-001 can effectively remove pathogens from the main target organs of infected chicks.

[0097] 3. Conclusion The induction culture supernatant of the mutant strain SJ-LI-LYS-001 showed significant preventive and therapeutic effects against *Riegeria duckweed* infection in chicks, increasing the survival rate of infected animals from 40% to 86.7%. It also effectively cleared the pathogen from the body, thereby inhibiting the pathogen's invasion of critical tissues such as the liver and brain, and protecting the integrity of tissue structures. These experimental results provide important in vivo experimental evidence for developing the mutant strain SJ-LI-LYS-001 into a safe, efficient, and cross-species biocontrol product for *Riegeria duckweed* infection.

[0098] 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 high-yield lactococcal mutant strain SJ-LI-LYS-001 of *Riebelella anatipestifer* phage lysin, characterized in that, Its classification name is Lactococcus lactis ( Lactococcus lactis It was deposited on January 9, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37338.

2. The use of the Lactococcus lactis mutant strain SJ-LI-LYS-001 according to claim 1 in the preparation of a medicament for the prevention and / or treatment of Riegeria duckweed infection in avian animals.

3. The application according to claim 2, characterized in that, The birds mentioned are young birds.

4. The application according to claim 3, characterized in that, The birds mentioned are chicks.

5. A method for expressing *Riebelella anatipestifer* phage lysin using the *Lactococcus lactis* mutant strain SJ-LI-LYS-001 as described in claim 1, characterized in that, The procedure includes the following steps: fermenting the Lactococcus lactis mutant strain SJ-LI-LYS-001 and then inducing it with Nisin.

6. The method according to claim 5, characterized in that, During the induction culture, the amount of Nisin added is 10-20 ng / mL; and / or During the induction culture, a 30% lactose aqueous solution was added at a rate of 30 mL / L / h.

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