Recombinant vector for expressing bacteriophage lyase as well as construction method and application of recombinant vector
The recombinant vector of phage lyase constructed using the Pichia pastoris expression system solves the stability problem of phage lyase under extreme environments, achieving efficient sterilization and wide application, suitable for high-temperature processed feed, oral preparations, and preservation of acidic foods.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUOLONG (NINGDE) BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-01-09
- Publication Date
- 2026-05-19
AI Technical Summary
Existing phage lysins are difficult to tolerate extreme gastrointestinal environments (such as low pH and digestive proteases) and high temperatures during feed processing when administered orally or in feed, resulting in insufficient stability and affecting their bactericidal effect.
A recombinant vector for expressing phage lyases was constructed using the Pichia pastoris expression system. The vector contained the phage lyase gene, multiple cloning site, promoter, and resistance selection marker. High-efficiency and stable expression of the lyase was achieved through Pichia pastoris expression, and lyases that could withstand high temperatures, strong acids, and pepsin and trypsin were screened out.
This study achieved highly efficient bactericidal activity of bacteriophage lysin against Clostridium perfringens and maintained stability under extreme conditions. It is suitable for high-temperature processed feed, oral preparations, and preservation of acidic foods, and has broad application prospects.
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Figure CN122060767A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biotechnology and microbial control, and in particular to a recombinant vector expressing bacteriophage lysin, its construction method, and its application. Background Technology
[0002] Clostridium perfringens is a widespread Gram-positive anaerobic spore-forming bacillus that is a major pathogen causing necrotic enteritis in animals, food poisoning in humans, and gas gangrene. Currently, controlling Clostridium perfringens mainly relies on antibiotics, but this leads to increasingly serious problems of drug resistance and drug residues. Phage lysins can specifically hydrolyze the peptidoglycan in bacterial cell walls, causing rapid bacterial lysis and death, and are less prone to inducing drug resistance, making them a potential candidate to replace antibiotics.
[0003] However, naturally occurring or conventionally expressed lyases face numerous challenges in practical applications, especially in oral or feed use, where they need to withstand extreme gastrointestinal environments (such as low pH and digestive proteases) and high temperatures during feed processing. Currently, most reported lyases have limitations in terms of stability, making the screening and acquisition of lyases that combine highly efficient bactericidal activity with excellent environmental tolerance of significant application value.
[0004] The Pichia pastoris expression system has become an important platform for the industrial production of exogenous proteins due to its ability to achieve correct folding, high-density fermentation, and secretory expression of eukaryotic proteins at a relatively low cost. Using this system to express phage lysins helps to obtain highly active products that are easy to process downstream. Summary of the Invention
[0005] In view of the above-mentioned shortcomings of current strains and products that inhibit or kill Clostridium perfringens, the present invention provides a recombinant vector expressing bacteriophage lysin, which has high efficiency lysing activity against Clostridium perfringens and is significantly resistant to high temperature, strong acid, and pepsin and trypsin.
[0006] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions: A recombinant vector for expressing a phage lyase, the recombinant vector for expressing a phage lyase being based on a Pichia pastoris expression system, the recombinant vector for expressing a phage lyase comprising a phage lyase gene, a multiple cloning site, a replication initiation site, a promoter, a terminator, an α-factor signal peptide, and an resistance selection marker.
[0007] According to one aspect of the present invention, the phage lysin gene is one of the following enzyme genes: CpLys849, CpLys978, CpLys435, CpLys008, CpLys359, CpLys844, CpLys484, CpLys948, CpLys128, CpLys702, CpLys515, and CpLys445D1.
[0008] According to one aspect of the present invention, the phage lysin gene sequence is shown in SEQ ID NO.1-SEQ ID NO.12.
[0009] According to one aspect of the invention, the multiple cloning site is a SmaI and an EcoRI / NotI multiple cloning site.
[0010] According to one aspect of the invention, the promoter is an AOX1 promoter, a TEF1 promoter, and an EM7 promoter.
[0011] According to one aspect of the invention, the resistance screening marker is BleoR resistance.
[0012] A method for constructing a recombinant vector expressing a phage lysin, the method comprising: The gene fragment was amplified, ligated with an enzyme-digested vector, transformed into competent cells, and cultured. Successful strains were screened, cultured, and plasmids were extracted. The successfully constructed recombinant plasmid was digested with enzymes and transformed into competent cells of Pichia pastoris, and positive clone strains were screened. Successfully expressed bacterial strains were inoculated into culture medium for culture and fermentation.
[0013] According to one aspect of the present invention, the enzyme-digested vector is the Pichia pastoris secretory expression vector pPICZαA, the vector is double-digested with EcoRI / NotI restriction endonucleases, and the competent cells are Escherichia coli TOP10.
[0014] According to one aspect of the invention, the strain that was successfully tested is a strain that can survive and reproduce on a resistant culture medium.
[0015] A recombinant vector expressing a phage lysin has applications in killing Clostridium perfringens and as a novel feed additive to prevent necrotic enteritis in poultry, as a food preservative to control Clostridium perfringens contamination in meat, and as a therapeutic drug for local or systemic infections.
[0016] Advantages of implementing this invention: (1) Highly efficient and specific bactericidal effect: The lysin screened in this invention has a highly efficient and rapid lysing effect on Clostridium perfringens; (2) Excellent environmental stability: This invention is the first to obtain phage lysins (Lys-05 and Lys-10) that can simultaneously withstand high temperature of 90℃, strong acid of pH 2.5 and high concentration of pepsin and trypsin through systematic tolerance screening. This gives them outstanding application advantages in scenarios such as feed that needs to be processed at high temperature, preparations that need to be administered orally and preservation of acidic food. (3) Advantages of the expression system: The Pichia pastoris expression system was used to realize the soluble secretion expression of the lysin. The fermentation process is mature, which is convenient for large-scale construction. The product is free of endotoxins and has high safety. (4) Broad application prospects: The extreme environment lyase provided by this invention can be used directly or after formulation to develop new feed additives to prevent necrotic enteritis in poultry, food preservatives to control Clostridium perfringens contamination in meat, or as a therapeutic drug for local or systemic infections, and has broad application prospects. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 This is a schematic diagram showing the initial screening results of the antibacterial activity of 12 recombinant lyases against Clostridium perfringens in this invention. Figure 2 The image shows the bactericidal effect of the seven active lysins CpLys849, CpLys978, CpLys435, CpLys484, CpLys128, CpLys702, and CpLys515 in this invention. Figure 3 This is a comparison chart of the relative residual activity of seven active lysins (CpLys849, CpLys978, CpLys435, CpLys484, CpLys128, CpLys702, and CpLys515) after high-temperature treatment in this invention. Figure 4 This is a comparison chart of the relative residual activity of seven active lysins (CpLys849, CpLys978, CpLys435, CpLys484, CpLys128, CpLys702, and CpLys515) after low pH treatment in this invention. Figure 5This is a comparison chart of the relative residual activity of five lysins (CpLys849, CpLys978, CpLys435, CpLys702, and CpLys515) after treatment with pepsin in this invention. Figure 6 This is a comparison chart of the relative residual activity of five lysins (CpLys849, CpLys978, CpLys435, CpLys702, and CpLys515) after treatment with trypsin in this invention. Figure 7 This is a schematic diagram illustrating the construction of the recombinant expression vector pPICZαA-CpLys in this invention. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0020] Example 1 Constructing recombinant expression vectors EcoRI and NotI were used for enzyme digestion. The enzyme digestion reaction system is shown in Table 1 below: Table 1
[0021] Enzyme digestion at 37℃ for 1 hour.
[0022] The pPICzα empty vector was retained from the laboratory. The EcoRI / Not I-HF® and 10X rCutSmart™ Buffer are from NEB.
[0023] Genes CpLys849, CpLys978, CpLys435, CpLys008, CpLys359, CpLys844, CpLys484, CpLys948, CpLys128, CpLys702, CpLys515, and CpLys445D1 with EcoRI and NotI restriction sites at both ends can be synthesized, for example, by Sangon Biotech (Shanghai) Co., Ltd., which is readily available.
[0024] The target gene and the digested pPICzα vector were ligated using T4 DNA ligase. The ligation system used is shown in Table 2 below. Table 2
[0025] Store overnight at 4°C.
[0026] The T4 DNA ligase was derived from takara.
[0027] Take TOP10 competent E. coli cells stored at -80℃, thaw them slowly on ice for about 5-10 minutes, avoiding repeated freeze-thaw cycles, add 50-100 μL of thawed TOP10 competent E. coli cells to a sterile EP tube, and incubate on ice for 2 minutes. Add 1-5 μL of plasmid DNA (total volume not exceeding 1 / 10 of the competent cell volume) to the TOP10 of E. coli competent cells, gently tap the bottom of the tube to mix, and incubate on ice for 30 minutes; Immediately immerse the EP tube in a 42℃ water bath for 45-90 seconds, avoiding shaking. Once the heat shock is complete, immediately transfer it to an ice bath to cool for 2-3 minutes. Add 800-950 μL of antibiotic-free LB liquid medium to the EP tube and incubate at 37°C and 200 rpm for 1 hour with shaking. Take 100-200 μL of the revived bacterial culture and spread it evenly on an LB agar plate containing bleomycin. If the bacterial concentration is too high, centrifuge at 5000 rpm for 1 minute, discard part of the supernatant, resuspend, and then spread. Invert the plate and incubate it in a 37°C incubator for 12-16 hours, then observe the growth of single colonies.
[0028] Single colonies that tested positive were cultured and plasmids were extracted for PCR verification. The reaction system is shown in Table 3 below. Table 3
[0029] The sequences of 9k-F3 and 9k-R2 are shown in SEQ ID NO.1-2; The 2× Hieff® PCR Master Mix was purchased from Yisheng Biotechnology.
[0030] The PCR reaction procedure is as follows: pre-denaturation at 94 degrees Celsius for 10 minutes; denaturation at 94 degrees Celsius for 30 seconds, annealing at 58 degrees Celsius for 30 seconds, extension at 72 degrees Celsius for 2 minutes for a total of 30 cycles; and finally, final extension at 72 degrees Celsius for 5 minutes, and storage at 4 degrees Celsius.
[0031] Example 2
[0032] EcoRI and NotI were used for enzyme digestion, and the enzyme digestion reaction system is shown in Table 4 below: Table 4
[0033] The Sac I-HF® was purchased from NEB; Enzyme digestion at 37℃ for 1 hour.
[0034] Melt competent GS115 Pichia pastoris cells on ice, add 1 μg of enzyme-digested plasmid, mix, add to a pre-cooled electroporator, and let stand on ice for 5 min; The competent GS115 Pichia pastoris was electroporated under the following conditions: 1.5 kV, 400 Ω, 25 μF. Immediately after electroporation, add 1 ml of 1 M sorbitol solution and wash into a centrifuge tube; Add an equal volume of antibiotic-free YPD medium, incubate in a shaker at 30°C, and allow to recover for 2-3 hours; After a brief centrifugation, discard 800 μL of supernatant and plate the sample onto a YPD plate containing 200 μg / mL BleoR antibiotic. Single colonies will be visible in 2-3 days. The colonies that grow are streaked onto 1000 ug / mL BleoR-resistant YPD high-antibody plates for screening of positive clones; The successfully expressed strains were inoculated into 10 mL of BMGY medium and cultured at 30°C and 160 rpm for 24 h. The BMGY medium was washed off by centrifugation and resuspension with sterile water, then transferred to 50 mL of BMMY medium and fermented at 30°C and 160 rpm for 5 days, with 1% methanol added every 24 hours. The initial OD of fermentation was <1.
[0035] The single colonies that tested positive were cultured and plasmids were extracted for PCR verification. The reaction system is shown in Table 5 below: Table 5
[0036] The sequences of colourF1 and colourR1 are shown in SEQ ID NO.3-4; The 2× Hieff® PCR Master Mix was purchased from Yisheng Biotechnology.
[0037] The PCR reaction procedure is as follows: pre-denaturation at 94 degrees Celsius for 10 minutes; denaturation at 94 degrees Celsius for 30 seconds, annealing at 58 degrees Celsius for 30 seconds, extension at 72 degrees Celsius for 2 minutes for a total of 30 cycles; and finally, final extension at 72 degrees Celsius for 5 minutes, and storage at 4 degrees Celsius.
[0038] Example 3 Lysase activity and tolerance experiment Antibacterial test: 5 mL of lysin solution was added to 25 mL of culture medium, and 5 μL of Clostridium perfringens was inoculated. The growth of the bacteria was observed within 8 hours. The schematic diagram of the initial screening results of antibacterial activity is shown below. Figure 1As shown.
[0039] Sterilization experiment: 32g of anaerobic liver broth culture medium was dissolved in 1000mL of distilled water by heating and stirring. After dispensing, a small amount of beef granules was added, and then the medium was covered with approximately 4mm of liquid paraffin. The mixture was autoclaved at 116℃ for 30-40 minutes and set aside. Clostridium perfringens was taken from cryopreservation tubes and inoculated into the anaerobic liver broth culture medium. The medium was cultured at 37°C under anaerobic conditions for 18-24 hours until the logarithmic phase (OD600=0.6). 5mL of treatment solution (experimental group / control group) + 25mL of bacterial suspension were incubated anaerobically at 37℃. The turbidity of the bacterial suspension was observed at 0, 1, 2, 3, 4, 6, 8, and 24 hours. A schematic diagram of the sterilization effects of seven active lyases (CpLys849, CpLys978, CpLys435, CpLys484, CpLys128, CpLys702, and CpLys515) is shown below. Figure 2 As shown.
[0040] Temperature tolerance: The enzyme solution was treated at 90℃ for 5 minutes, then immediately placed in ice water to stop tolerance. The lysin solution was then mixed with *Clostridium perfringens* in the logarithmic growth phase and cultured, with turbidity observed at different time points. A comparison of the relative residual activity of seven active lysins (CpLys849, CpLys978, CpLys435, CpLys484, CpLys128, CpLys702, and CpLys515) after high-temperature treatment is shown in the figure below. Figure 3 As shown.
[0041] pH tolerance: The pH of 5 mL of fermentation broth was adjusted to 2.5 with 6 mol / L hydrochloric acid solution and treated for 1 h (simulating gastric acid environment). Then, an equal volume of 6 mol / L sodium hydroxide solution was added to adjust the pH of the fermentation broth back to its original pH. The lyase solution was then mixed with *Clostridium perfringens* in the logarithmic growth phase and cultured according to the prescribed steps. Turbidity of the bacterial culture was observed at different time points. The relative activity residues of seven active lyases (CpLys849, CpLys978, CpLys435, CpLys484, CpLys128, CpLys702, and CpLys515) after low pH treatment are compared as shown in the figure. Figure 4 As shown.
[0042] Resistance to pepsin and trypsin degradation: A hydrochloric acid solution containing 100 U / mL pepsin at pH 2.5 was prepared. Both the experimental and control groups were treated with this solution for 1 hour to tolerate the enzymes, then immediately reconstituted with sodium hydroxide solution. The lysin solution was then mixed with *Clostridium perfringens* in the logarithmic growth phase and cultured, with turbidity observed at different time points. The relative residual activity of five lysins (CpLys849, CpLys978, CpLys435, CpLys702, and CpLys515) after pepsin treatment is shown in the figure below. Figure 5 As shown.
[0043] Antitrypsin resistance: A phosphate-buffered saline solution containing 100 U / mL trypsin at pH 6.8 was prepared. Each group was treated with this solution for 1 hour to tolerate the lysin. Then, the lysin solution was mixed with *Clostridium perfringens* in the logarithmic growth phase and cultured. Turbidity of the bacterial culture was observed at different time points. The relative residual activity of the five lysins (CpLys849, CpLys978, CpLys435, CpLys702, and CpLys515) after trypsin treatment is compared in the following figure. Figure 6 As shown.
[0044] Advantages of implementing this invention: (1) Highly efficient and specific bactericidal effect: The lysin screened in this invention has a highly efficient and rapid lysing effect on Clostridium perfringens; (2) Excellent environmental stability: This invention is the first to obtain phage lysins (Lys-05 and Lys-10) that can simultaneously withstand high temperature of 90℃, strong acid of pH 2.5 and high concentration of pepsin and trypsin through systematic tolerance screening. This gives them outstanding application advantages in scenarios such as feed that needs to be processed at high temperature, preparations that need to be administered orally and preservation of acidic food. (3) Advantages of the expression system: The Pichia pastoris expression system was used to realize the soluble secretion expression of the lysin. The fermentation process is mature, which is convenient for large-scale construction. The product is free of endotoxins and has high safety. (4) Broad application prospects: The extreme environment lyase provided by this invention can be used directly or after formulation to develop new feed additives to prevent necrotic enteritis in poultry, food preservatives to control Clostridium perfringens contamination in meat, or as a therapeutic drug for local or systemic infections, and has broad application prospects.
[0045] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A recombinant vector expressing a phage lysin, wherein the recombinant vector expressing the phage lysin is based on a Pichia pastoris expression system, characterized in that, The recombinant vector expressing phage lyase includes a phage lyase gene, a multiple cloning site, a replication initiation site, a promoter, a terminator, an α-factor signal peptide, and an resistance selection marker.
2. The recombinant vector expressing bacteriophage lysin according to claim 1, characterized in that, The phage lysin gene is one of the following: CpLys849, CpLys978, CpLys435, CpLys008, CpLys359, CpLys844, CpLys484, CpLys948, CpLys128, CpLys702, CpLys515, and CpLys445D1.
3. The recombinant vector expressing bacteriophage lysin according to claim 2, characterized in that, The phage lysin gene sequences are shown in SEQ ID NO.1-SEQ ID NO.
12.
4. The recombinant vector expressing bacteriophage lysin according to claim 1, characterized in that, The multiple cloning sites are SmaI and EcoRI / NotI multiple cloning sites.
5. The recombinant vector expressing bacteriophage lysin according to claim 1, characterized in that, The promoters are AOX1 promoter, TEF1 promoter and EM7 promoter.
6. The recombinant vector expressing bacteriophage lysin according to claim 1, characterized in that, The resistance screening marker is BleoR resistance.
7. A method for constructing a recombinant vector expressing a phage lysin according to any one of claims 1-6, characterized in that, The construction method includes: The gene fragment was amplified, ligated to an enzyme-digested vector, transformed into competent cells, and cultured. Successful strains were screened, cultured, and plasmids were extracted. The successfully constructed recombinant plasmid was digested with enzymes and transformed into competent cells of Pichia pastoris, and positive clone strains were screened. Successfully expressed bacterial strains were inoculated into culture media for culture and fermentation.
8. The recombinant vector expressing bacteriophage lysin according to claim 7, characterized in that, The digested vector is the Pichia pastoris secretory expression vector pPICZαA, which is double-digested with EcoRI / NotI restriction endonucleases, and the competent cells are Escherichia coli TOP10.
9. The recombinant vector expressing bacteriophage lysin according to claim 7, characterized in that, The strains that were successfully tested are those that can survive and reproduce on resistant culture media.
10. The application of a recombinant vector expressing a phage lysin in killing Clostridium perfringens and as a novel feed additive to prevent necrotic enteritis in poultry, as a food preservative to control Clostridium perfringens contamination in meat, and as a therapeutic drug for local or systemic infections.