Vibrio parahaemolyticus phage in vivo lysin LysC75 and its application
By developing the in vivo lysozyme LysC75 of Vibrio parahaemolyticus phage, a bactericide was prepared, solving the problem of Vibrio parahaemolyticus control and achieving a highly efficient and safe bactericidal effect, which is suitable for the food industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2026-03-10
AI Technical Summary
Current technologies lack effective methods to control Vibrio parahaemolyticus, and the use of antibiotics may lead to the emergence of antibiotic-resistant bacteria, affecting food safety.
We developed LysC75, a bacteriophage lysin from Vibrio parahaemolyticus, which exhibits high bactericidal efficacy and specificity by lysing the host cell wall and can be used to prepare bactericides.
This bactericide can significantly reduce the concentration of Vibrio parahaemolyticus, with a sterilization rate of over 90%. It is highly specific to Vibrio parahaemolyticus, has high safety, and does not affect the structure and sensory characteristics of food.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of sterilization technology, in particular to Vibrio parahaemolyticus phage endolysin LysC75 and application thereof. BACKGROUND
[0002] Vibrio parahaemolyticus is a gram-negative bacterium without spores, and contaminated and undercooked seafood is the main transmission route of Vibrio parahaemolyticus, which can cause abdominal pain, vomiting, diarrhea and other diseases in consumers.
[0003] At present, there are few alternatives for controlling Vibrio parahaemolyticus in aquaculture, including some disinfectants and a few legally permitted antibiotics. The use of antibiotics is a common practice for treating diseases, but the overuse of antibiotics can lead to the emergence of antibiotic-resistant bacteria, resulting in drug-resistant bacteria even multiple drug-resistant "superbugs" posing a huge threat to food safety. The study of phages has once again attracted the attention of researchers around the world. However, bacteria can also develop resistance to phages, which limits the application of phages. Therefore, some researchers focus on endolysins produced during the lysis of host cells by virulent phages. The bactericidal property of endolysin as the main active substance has many advantages such as easy to be directed to modify by gene editing and not to be neutralized by antibodies, etc., which can safely and efficiently reduce the contamination level of specific pathogenic bacteria in food and will not have harmful effects on the structure and sensory properties of food, and has the potential to be applied in the food industry.
[0004] Endolysins are enzymes used by phages to lyse the host cell wall in the final stage of the lytic cycle. Phages containing double-stranded DNA can lyse the peptidoglycan layer by producing endolysins in the final stage of the lytic cycle. This mechanism represents a promising enzyme-based antibacterial effect. Endolysin as the main active substance of bactericides has the following advantages in controlling bacterial pathogens in food: (i) rapid reduction of target bacteria; (ii) expanded antibacterial spectrum compared to phages; (iii) reduced resistance rate; and (iv) improved safety for food applications without any toxins and. Many studies related to endolysins show that endolysins can kill Escherichia coli O157:H7, Listeria monocytogenes, Campylobacter jejuni, Salmonella and Pseudomonas. So far, a representative phage endolysin recombinant protein P128 is in the late stage of preclinical drug development for the local prevention and treatment of Staphylococcus skin infections. However, there is still no commercially available Vibrio phage endolysin bactericide.
[0005] In summary, it is of great value to explore a new endolysin with significant bacteriostatic effect for the prevention and control of Vibrio parahaemolyticus. SUMMARY
[0006] Based on the technical problems existing in the background art, this invention proposes the in vivo lysin LysC75 for Vibrio parahaemolyticus and its application, which can effectively inhibit the growth of Vibrio parahaemolyticus, has strong specificity for Vibrio parahaemolyticus and high lysis efficiency, and can be used for the prevention and control of Vibrio parahaemolyticus.
[0007] The present invention proposes a endolysin LysC75 for Vibrio parahaemolyticus phage, the amino acid sequence of which is shown in SEQ ID NO.2.
[0008] The present invention proposes a DNA comprising a gene encoding the above-mentioned Vibrio parahaemolyticus phage lysozyme LysC75, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0009] The present invention proposes an expression vector comprising the aforementioned DNA.
[0010] The present invention proposes a microorganism that is transformed using the above-mentioned expression vector.
[0011] The present invention proposes the application of the above-mentioned Vibrio parahaemolyticus phage lysozyme LysC75 in bactericides.
[0012] The present invention proposes an enzyme preparation comprising the above-mentioned Vibrio parahaemolyticus phage lysozyme LysC75.
[0013] Beneficial technical effects of the present invention:
[0014] This invention provides a bactericide with Vibrio parahaemolyticus endosomalin as the main active substance. This bactericide effectively inhibits the growth of Vibrio parahaemolyticus, exhibiting high specificity and lysis efficiency against the bacteria, and can be used for the control of Vibrio parahaemolyticus. The bactericidal effect is dose-dependent; a 1 mg / mL concentration of the bactericide reduces the Vibrio parahaemolyticus concentration from 10 mg / mL to 10 mg / mL within 120 minutes. 8 CFU / mL dropped to 10 5 A bactericide at a concentration of CFU / mL and 0.3 mg / mL can reduce bacterial concentration from 10... 8 CFU / mL dropped to 10 7 CFU / mL. A concentration of 1 mg / mL of this fungicide is effective against ≤10 3 The bactericidal effect of CFU / mL on Vibrio parahaemolyticus is 100%, and it is effective against 10... 6 -10 4 The sterilization rate of CFU / mL strains can reach over 90%, and for strains of 10 9 -10 6 The sterilization rate of CFU / mL strains can reach over 90%.
[0015] This invention screened and isolated a new virulent bacteriophage resistant to multidrug-resistant bacteria from wastewater in a wastewater treatment plant. Through research on its performance, it was found that the bacteriophage has a broad host spectrum, good temperature and pH stability, and good antibacterial effect. Attached Figure Description
[0016] Figure 1 This is a phage plaque diagram of phage C75 proposed in this invention.
[0017] Figure 2 This is an electron microscope image of the bacteriophage C75 proposed in this invention.
[0018] Figure 3 This is a schematic diagram of the infection multiple of bacteriophage C75 proposed in this invention.
[0019] Figure 4 This is a schematic diagram of the one-step growth curve of bacteriophage C75 proposed in this invention.
[0020] Figure 5 The present invention provides a VIRIDIC heatmap of phage C75.
[0021] Figure 6 This is a heatmap of genome comparison of different bacteriophages using Easyfig proposed in this invention.
[0022] Figure 7 This is the phylogenetic tree of the endosomalin LysC75 proposed in this invention.
[0023] Figure 8 The recombinant plasmid map proposed in this invention.
[0024] Figure 9 This is an electrophoresis image of the purified protein of the recombinant phage in vivo lysin LysC75 proposed in this invention.
[0025] Figure 10 This is a graph showing the antibacterial activity assay of the phage in vivo lysin LysC75 proposed in this invention.
[0026] Figure 11 This is a diagram showing the bactericidal effect of the endolysin bactericide proposed in this invention at 0 min.
[0027] Figure 12 The image shows the bactericidal effect of the endolytic bactericide proposed in this invention after 30 minutes.
[0028] Figure 13 This is a diagram showing the bactericidal effect of the endolytic bactericide proposed in this invention after 60 minutes.
[0029] Figure 14 This is a diagram showing the bactericidal effect of the endolytic bactericide proposed in this invention after 90 minutes.
[0030] Figure 15 The image shows the bactericidal effect of the endolytic bactericide proposed in this invention after 120 minutes.
[0031] Figure 16 The diagram shows the bactericidal effect of the endolytic bactericide proposed in this invention at different times and concentrations.
[0032] Figure 17 The diagram shows the bactericidal effect of the endosomalin bactericide proposed in this invention on bacteria of different concentrations.
[0033] Figure 18 The temperature stability of the endolytic bactericide proposed in this invention.
[0034] Figure 19 The pH stability of the endosomal bactericide proposed in this invention. Detailed Implementation
[0035] The present invention will be further explained below with reference to specific embodiments.
[0036] The bacteriophage C75 in this invention is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC M 2025326, deposit date February 28, 2025, and classification name: Vibrio parahaemolyticus vB_VpaP_XY75.
[0037] The strains in the C75 host spectrum of phage in Table 1 are from our laboratory collection.
[0038] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Example 1
[0039] Isolation and purification of bacteriophages
[0040] Water sample treatment
[0041] Wastewater samples were collected from a wastewater treatment plant. 10,000g of the sample was centrifuged for 10 minutes to remove large solid particles and some bacteria. The supernatant was collected and vacuum-filtered through a 0.45mm aqueous filter membrane into a clean, sterile Erlenmeyer flask. MgSO4 was added to a final concentration of 50mM, stirred thoroughly, and allowed to stand for 15-20 minutes. The mixture was then vacuum-filtered, the supernatant discarded, and the filter membrane collected. The membrane was cut into small pieces and placed in a beaker containing an appropriate amount of eluent. The mixture was sonicated for 4-5 minutes to elute the bacteriophage particles from the membrane. The mixture was then filtered through a 0.45mm filter into another dry, sterile Erlenmeyer flask. This liquid is the bacteriophage mixture and should be stored at 4°C for later use.
[0042] Under aseptic conditions, inoculate Vibrio parahaemolyticus into 5 mL of TSB medium and incubate overnight. Inoculate the activated bacterial culture at a 1% inoculum into fresh 5 mL TSB medium and incubate until the logarithmic growth phase. Mix 500 mL of the phage mixture, 500 mL of double-strength TSB medium (containing 2 mM CaCl2 to a final concentration), and 50 mL of the logarithmic growth phase bacterial culture thoroughly and incubate at 37°C with shaking for 8 h. Centrifuge the culture at 4000 g for 15-20 min and filter through a 0.45 mm filter into sterile centrifuge tubes to obtain the phage lysis buffer, which should be stored at 4°C for later use.
[0043] Phage purification
[0044] Pour 1.5% TSA (tryptic soy agar) onto a clean, sterile culture dish. Add 100 mL of bacterial culture in the logarithmic growth phase and 100 mL of phage lysis buffer to 5 mL of 0.7% TSB-Ca soft agar, mix thoroughly, and spread evenly onto the surface of dry 1.5% TSB agar. Incubate at 37°C for 8 hours to obtain clearly visible phage plaques. Use a sterile pipette tip to pick up a single transparent phage plaque, dissolve it in an appropriate amount of SM buffer, mix thoroughly, centrifuge at 4000g for 15-20 min, collect the supernatant, and filter the supernatant through a 0.45 mm filter to obtain the phage purification solution. Repeat the purification process multiple times until the phage plaques are uniformly transparent. Figure 1 ).
[0045] Phage proliferation and concentration
[0046] Take 1 mL of TSB-Ca broth culture medium into a sterile centrifuge tube, add 20 mL of bacterial culture in the logarithmic growth phase and 20 mL of phage purification solution, and incubate at 37℃ and 200 rpm for 8 h. Centrifuge at 4000g for 15-20 min, filter through a 0.45 mm filter into a new sterile centrifuge tube, which is the phage proliferation solution. Add 0.5M NaCl and 10% polyethylene glycol 8000 to the proliferation solution, and incubate overnight at 4℃ to allow the phage particles to precipitate. Centrifuge at 12000g for 10 min, discard the supernatant, collect the precipitate, and redissolve the precipitate in SM buffer to obtain the phage concentrate. Store at 4℃ for later use. (For long-term storage, add 30% glycerol to the final concentration and store at -20℃.) Determine its potency using the double-layer agar plate method. Example 2
[0047] Observation of bacteriophage morphology
[0048] The purified phage suspension was dropped onto a copper grid. After 3-5 minutes, the liquid was absorbed with filter paper. A drop of PTA was added, and staining was performed for 2-3 minutes. Excess liquid was then absorbed, and the phage was air-dried. Observation was then performed using a field emission transmission electron microscope. Phage C75 belongs to the Myoviridae family, with a head of approximately 40 mm and a tail of approximately 62 mm. Figure 2 ). Example 3
[0049] Determination of the host spectrum of bacteriophage C75
[0050] Spread 1.5% TSB agar evenly onto a dry, sterile petri dish and allow it to dry. Add 100 mL of bacterial culture in the logarithmic growth phase to 5 mL of 0.4% TSB, mix well, spread evenly onto the dried plate, and allow it to air dry until the soft agar solidifies. Add 2 mL of phage culture to the soft agar using the spotting method, allow it to air dry, and then incubate at 37°C for 4-6 hours. The lysis effect is divided into clear (+) and no plaques (-) in the spotting area, and the results are shown in Table 1. Host spectrum determination of C75 was performed using 27 bacterial strains. The results showed that the bactericidal rate of phage C75 was 44%, and it had no infectivity against non-parahaemolytic Vibrio strains, indicating that it has good specificity.
[0051] Table 1. Results of host spectrum determination for bacteriophage C75
[0052] Serial number Strain name Bacterial species Lysis effect 1 ATCC 17802 - ]]> 2 GD91 + 3 GD103 + 4 GD71 + 5 GD78 - ]]> 6 GD81 - ]]> 7 GD62 + 8 GD75 + 9 GD74 + 10 GD89 + 11 GD80 + 12 GD83 + 13 GD73 - ]]> 14 99-1 - ]]> 15 66-1 - ]]> 16 124-1 - ]]> 17 84-1 + 18 163-1 - ]]> 19 234-2 + 20 234-1 - ]]> 21 84-5 + 22 ATCC 13076 - ]]> 23 ATCC 19111 - ]]> 24 ATCC 51329 - ]]> 25 ATCC 19433 - ]]> 26 ATCC 14028 - ]]> 27 ATCC 49128 - ]]> Example 4
[0053] Determination of the optimal multiplicity of infection by bacteriophage
[0054] The bacterial cells were cultured to the pre-log phase to achieve a bacterial concentration of 10. 8 CFU / mL, phage fluid and host bacterial culture were added at multiples of infection ratios of 100:1, 10:1, 1:1, 1:10, 1:100, 1:1000, and 1:10000. The mixtures were incubated at 37°C and 200 rpm for 4 hours, centrifuged at 12000 rpm for 10-15 minutes, filtered through a 0.45 µm filter, and titer was determined using the double-layer agar plate method. The infection that produces the highest possible multiple of infection is considered optimal.
[0055] like Figure 3 As shown, the potency is highest at MOI=1:100, reaching 8.1×10⁻⁶. 10 pfu / mL, meaning the optimal MOI for phage C75 is 1:100. Example 5
[0056] Determination of one-step growth curve of bacteriophage
[0057] The bacterial cells were cultured to the pre-log phase to achieve a bacterial concentration of 10. 8CFU / mL was added to the host bacteria and phage culture medium at an optimal multiplicity of infection (MOI) of 1:100, and incubated at 37°C with shaking. Samples were taken at 0 min, every 5 min for the first 20 min, and every 10 min thereafter. The samples were centrifuged at 12000 rpm for 30 s, filtered through a 0.45 mm filter, and the phage titer was determined at each time point. A one-step growth curve was plotted with infection time on the x-axis and phage titer on the y-axis.
[0058] like Figure 4 As shown, 0-5 min is the adsorption period of phage C75, 10-20 min is the lysis period of phage C75, and 30-120 min is the stationary period of phage C75. Example 6
[0059] Phage genome sequencing and analysis
[0060] Add DNase I and RNase A to 500 mL of the phage concentrate from Example 1, and incubate at 37°C for 60 min (inverting every 30 min). Add 10% SDS, proteinase K, and EDTA, and incubate at 65°C for 60 min (inverting every 30 min). Add an equal volume of Tris-saturated phenol, vortex for 30 s, centrifuge at 12000 g for 5 min, and transfer the supernatant to a new centrifuge tube. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), vortex for 30 s, centrifuge at 12000 g for 5 min, and transfer the supernatant to a new centrifuge tube. Extract with an equal volume of chloroform, vortex for 30 s, centrifuge at 12000 g for 5 min, and transfer the supernatant to a new centrifuge tube. Repeat this operation twice. Add an equal volume of isopropanol, mix well, incubate at -20°C for 30 min, centrifuge at 12000 g for 20 min at 4°C, and collect the precipitate. Wash the precipitate with 200 mL of 70% ethanol, centrifuge at 12000 g for 5 min, discard the supernatant, and retain the precipitate. Repeat twice. Dry the DNA precipitate at room temperature, add 20 mL of sterile water preheated to 65 °C, and store at -20 °C for later use.
[0061] Illumina sequencing results show that phage C75 is a linear, dsDNA phage. The complete genome sequence of phage C75 is 44,860 bp in length, with a GC content of 48.68% and containing 50 coding sequences (CDS). Compared with virulence databases and antibiotic resistance gene databases, phage C75 does not contain virulence genes or antibiotic genes, indicating that it can be safely used to control Vibrio parahaemolyticus. Figure 5The VIRIDIC heatmap of phage C75 shows that phage C75 has 100% similarity to Vibrio phage vB_VpaP_MGD1 (coverage 96.03%) and 98% similarity to Vibrio phage vB_VpaP_1701 (coverage 95.28%).
[0062] Figure 6 A comparative heatmap of the genomes of different bacteriophages was generated using Easyfig. Bacteriophage C75 was predicted to contain 50 ORFs but no tRNA. Of these, 26 ORFs were annotated as functional proteins, while the rest were annotated as putative proteins without tRNA. The functional proteins of the bacteriophage were divided into five modules: DNA metabolism, lysis, packaging, structure, and additional functions. Example 7
[0063] Endolysin protein expression
[0064] After sequencing its whole genome and uploading the results to the NCBI database, the original coding nucleotide sequence of the phage lysin LysC75 was obtained by BLAST alignment, as shown in SEQ ID NO 1:
[0065] ATGCTAATTAAAGACACTGCGAATTTCAAGATTACGAAGTTCGCGTGTCAGCATTGTGGGGCTTTGAAACTAGACCTAGCCCTGCTTATGCTGGTACAAATGCTACGGGAGCACTTCGGAGAACCGCTAAAGGTTGAATCCGGTTATCGCTGCCCTGTACACAATAAAGCCGTAGGCGGTGCTGAGGACTCTCGTCACTTACATGGT GATGCAGTGGACTTGCACTTGCTGAACAAAGACCGGGGGAACTTCCAGAAGCTCCAGAAGCTGTACGACACGGCTTTAGCTCTGAACCCTAACGGTGGCGTCGGTCTGTACGACTGGGGTGTACACGTTGATACACGCGGTGAGAAAGCCCGTTGGGATTACCGCTCTGATAAATACAAAGAAGTAATGGGGAAAATGGATGTCTGA
[0066] The amino acid sequence corresponding to the phage lysin LysC75 is shown in SEQ ID NO.2:
[0067] MLIKDTANFKITKFACQHCGALKLDLALLMLVQMLREHFGEPLKVESGYRCPVHNKAVGGAEDSRHLHGDAVDLHLLNKDRGNFQKLQKLYDTALALNPNGGVGLYDWGVHVDTRGEKARWDYRSDKYKEVMGKMDV
[0068] like Figure 7 As shown in the phylogenetic analysis of endolysins, the lyases in phage endolysin LysC75 and vB_VpaP_MGD1 have high homology.
[0069] The recombinant plasmid PGEX-4T-1-LysC75 was obtained by adding the artificially synthesized gene to the EcoRI and XhoI restriction sites of the PGEX-4T-1 plasmid. This plasmid was then introduced into *E. coli* BL21 competent cells to obtain positive clones containing the PGEX-4T-1-LysC75 plasmid. The specific procedures are as follows:
[0070] Using the phage endosomal genome as a template, a gene of approximately 500 bp was successfully amplified using primers. The PCR product was consistent with the expected target fragment (414 bp). The plasmid pGEX-4T-1 and the purified PCR product were double-digested with XholI and EcoRI restriction endonucleases. After verifying the correctness of the target band by 1.5% agarose gel electrophoresis, the plasmid and target gene with identical sticky ends were obtained. The obtained vector with sticky ends and the target gene were ligated using T4 ligase. The ligated recombinant plasmid was then transformed into competent BL21 cells. Sequencing results showed that the nucleotide sequence was correct, indicating successful construction of the recombinant plasmid. The recombinant plasmid map is shown below. Figure 8 As shown.
[0071] The primers are as follows:
[0072] Upstream primer (SEQ ID NO 3): CCCTCGAGTCAGACATCCATTTTCCCC
[0073] Downstream primer (SEQ ID NO 4): CGGAATTCATGCTAATTAAAGACACTGCGA
[0074] The positive clone strain was cultured and its protein expression was induced to obtain the Vibrio parahaemolyticus phage lysin LysC75, which was then enriched and concentrated. IPTG was used to induce the expression of the vector fusion protein, as detailed below:
[0075] (1) The recombinant bacterial culture solution was inoculated at a ratio of 1% into LB broth medium containing Amp (final concentration 50 µg / mL), and the recombinant bacteria were cultured at 37°C and 200 rpm until OD. 600 =0.6-0.8; then add isopropyl thiogalactoside (IPTG) to the cultured recombinant bacteria at a final concentration of 1mM, and induce at 16℃ and 200rpm for 12-16h.
[0076] (2) Centrifuge the bacterial culture at 4℃ and 10000g for 10 min, discard the supernatant, and resuspend the bacterial cells in cell lysis buffer. Add 1mM benzyl sulfonyl fluoride (PMSF) before ultrasonic disruption to prevent protein denaturation. Disrupt the bacterial cells with ultrasound (power: 70W, 4s on, 8s off, 10 min total). Centrifuge the disruption solution at 4℃ and 12000g for 30 min, collect the supernatant, which is the crude protein.
[0077] Purify the crude protein using the Mag-Beads GST fusion protein purification magnetic beads according to the instructions. Prepare SDS-PAGE gels (5% stacking gel, 12% separating gel); mix the flow-through, wash, and elution samples of the purified protein with the protein loading buffer, boil at 100°C for 3-5 min, centrifuge at 12000g for 2-5 min, and load the supernatant for electrophoresis. After electrophoresis, stain the protein with Coomassie Brilliant Blue, and then destain three times with distilled water.
[0078] like Figure 9 The SDS-PAGE spectrum shown shows lanes 1-7, indicating that impurities were removed after four washes. Lane 8 is the eluent for endolysin purified by magnetic beads; the target band size is 42 kDa, and only one band appears, proving that endolysin expression and purification were successful.
[0079] Add the purified protein solution to a dialysis bag at a protein-to-dialysis buffer ratio of 1:10. Place the bag in the dialysis buffer and incubate at 4°C with a magnetic stirrer for 12 hours. Centrifuge the dialyzed protein at 12,000 rpm for 20 minutes at 4°C, remove the precipitate, and concentrate the supernatant using a 30,000 kDa ultrafiltration tube at 4°C and 3000 g. Example 8
[0080] The GD83 bacterial culture was cultured to 10... 8 Centrifuge at 8000g for 10 min with CFU / mL, discard the supernatant, wash the precipitate with sterile physiological saline buffer, repeat the washing twice, and resuspend the bacterial cells with an appropriate amount of physiological saline buffer to achieve an OD value of [insert value here]. 600=1. Six treatment groups were designed based on the resuspension: ① no addition; ② addition of 5 mM EDTA; ③ addition of 1 mg / mL lysozyme solution; ④ addition of 1 mg / mL lysozyme solution and 5 mM EDTA external permeation agent; ⑤ addition of 0.1 mg / mL lysozyme solution; ⑥ addition of 0.1 mg / mL lysozyme solution and 5 mM EDTA external permeation agent. The mixtures were incubated at 37℃ for 5 min, 30 min, 60 min, 90 min, and 120 min, and the OD of the mixtures was measured using a UV spectrophotometer. 600 , used to reflect bacterial concentration.
[0081] The results are as follows Figure 10 As shown, the difference between using EDTA alone and the control group was small; endolysin alone could not effectively kill Vibrio parahaemolyticus, while the bactericide was significantly more effective than either EDTA alone or endolysin alone. Endolysin showed bactericidal effect within 5 minutes, with the highest bacterial killing efficiency between 5 and 30 minutes. The bactericidal effect was concentration-dependent; adding a final concentration of 1 mg / mL endolysin was significantly more effective than adding a final concentration of 0.1 mg / mL endolysin, indicating that a higher concentration of endolysin in the formulation resulted in better bactericidal effect.
[0082] Cultivate GD80 bacterial culture to 10 8 Centrifuge at 8000g for 10 min to allow bacterial cells to precipitate at the bottom of the centrifuge tube. Discard the supernatant, wash the precipitate twice, and resuspend the bacterial cells in an equal volume of sterile physiological saline to achieve a bacterial concentration of 10⁻⁶ CFU / mL. 8 CFU / mL. Different concentrations of bactericide were added to the bacterial culture, with sterile physiological saline buffer added as a negative control. The mixtures were incubated at 37°C for 0 min, 30 min, 60 min, 90 min, and 120 min, respectively.
[0083] Figure 11-15 The images show the sterilization effects at concentration gradients of 1-0.1 mg / mL for 0 min, 30 min, 60 min, 90 min, and 120 min, respectively. Figure 16 for Figure 11-15 The experiment corresponds to the number of bactericidal effects. The bactericidal effect is concentration-dependent, and the bactericidal efficiency is highest within 30 minutes. Under 1 mg / mL bactericidal agent conditions, the concentration of Vibrio parahaemolyticus can be reduced from 10... 8 CFU / mL dropped to 10 6 The best bactericidal effect is achieved at CFU / mL, with 2 hours, ultimately reducing the Vibrio parahaemolyticus concentration from 10... 8 CFU / mL dropped to 10 5CFU / mL. Among bactericides in the 0.1-1 mg / mL endosomalin concentration range, higher concentrations are more effective. The minimum effective concentration is 0.3 mg / mL; a bactericide at this concentration can reduce the concentration of Vibrio parahaemolyticus from 10 CFU / mL to 10 CFU / mL within 120 minutes. 8 CFU / mL to 10 7 CFU / mL.
[0084] Cultivate GD80 bacterial culture to 10 8 Centrifuge at 8000g for 10 min to allow bacterial cells to precipitate at the bottom of the centrifuge tube. Discard the supernatant and wash the precipitate twice to achieve a bacterial concentration of 10. 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 CFU / mL of lysin and EDTA were added to the bacterial culture, with PBS buffer added as a negative control. The mixtures were incubated at 37°C for 2 hours, and the bacterial counts were performed using the PCA plate count method.
[0085] Figure 17 This image shows the bactericidal effect of a bactericide on bacterial solutions of different concentrations. The bacterial concentration is at 10... 3 At CFU / mL and below, the bactericide achieves 100% clearance, effectively combating Vibrio parahaemolyticus infections in the environment. Even under high bacterial concentrations (10⁻⁶ CFU / mL), the bactericide's effectiveness is enhanced. 9 -10 4 Even with a concentration of CFU / mL, the bactericide's sterilization rate can still reach over 90%. Example 9
[0086] Stability at different temperatures and pH levels was determined. GD80 bacterial culture was cultured to 10⁸ CFU / mL, centrifuged at 8000g for 10 min to allow bacterial cells to precipitate at the bottom of the centrifuge tube, the supernatant was discarded, and the precipitate was washed twice. 100 mL of 10 mg / mL LysC75 was placed in a 1.5 mL centrifuge tube and incubated at 4℃, 10℃, 20℃, 30℃, 37℃, 50℃, 60℃, and 70℃ for 1 h. After incubation, the tube was removed and cooled on ice, then added to the resuspended GD80 bacterial culture and incubated at 37℃ for 2 h. The concentration of GD80 bacteria was determined by plate counting. Take 100 mL of LysC75 with a concentration of 10 mg / mL into a 1.5 mL centrifuge tube, add different pH values (50 mM citrate buffer pH 3-6, 50 mM and 50 mM tris-HC1 buffer pH 7-9), incubate at 37 °C for 2 h, and determine the concentration of GD80 bacteria by plate counting.
[0087] The result is Figure 18, Figure 19 As shown, the bactericide's bactericidal effect remained almost unchanged after treatment at 4℃-37℃ for 1 hour. However, after treatment at 50℃ for 1 hour, the bactericidal rate decreased by 50%, and the potency decreased. After treatment at 60℃ and 70℃ for 1 hour, the bactericide was completely inactivated. The bactericide's bactericidal effect remained almost unchanged at pH 4-9, but it was completely inactivated below pH 5.
[0088] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. The scope of this application is defined by the appended claims and their equivalents, all of which should be included within the protection scope of this application.
Claims
1. A type of Vibrio parahaemolyticus ( Vibrio parahaemolyticus bacteriophage ( phage The application of endosomalin LysC75 in the preparation of bactericides, characterized in that, The amino acid sequence of the Vibrio parahaemolyticus phage endolysin LysC75 is shown as SEQ ID NO. 2; the bactericide is used for sterilization of Vibrio parahaemolyticus.