Vibrio parahaemolyticus bacteriophage endolysin LysC75 and application thereof

Abstract

The invention discloses vibrio parahaemolyticus bacteriophage endolysin LysC75 and application thereof. The amino acid sequence of the endolysin LysC75 is shown as SEQ ID NO. 2. The bactericide with the vibrio parahaemolyticus phage endolysin as the main active substance can efficiently split vibrio parahaemolyticus, the effective bactericidal concentration of the bactericide is larger than or equal to 0.2 mg / mL, and when the bactericide with the endolysin concentration being 1 mg / mL acts for 120 min, the high-concentration vibrio parahaemolyticus can be reduced from 10 < 8 > CFU / mL to 10 < 5 > CFU / mL. The sterilizing effect of the sterilizing agent with the concentration of 1 mg / mL on the vibrio parahaemolyticus being smaller than or equal to 10 < 3 > CFU / mL is 100%, the sterilizing rate of the sterilizing agent on 10 < 6 >-10 < 4 > CFU / mL bacterial strains can reach 90% or above, and the sterilizing rate of the sterilizing agent on 10 < 9 >-10 < 6 > CFU / mL bacterial strains can reach 90% or above. The bactericide has strong specificity and high cracking efficiency on vibrio parahaemolyticus, and can provide an important alternative strategy for prevention and control of drug-resistant vibrio parahaemolyticus.

Description

Technical Field

[0001] The present invention relates to the field of sterilization technology, and particularly to Vibrio parahaemolyticus bacteriophage endolysin LysC75 and its application. Background Art

[0002] Vibrio parahaemolyticus is a Gram-negative bacterium without spores. Contaminated and undercooked seafood is the main transmission route of Vibrio parahaemolyticus, which can cause consumers to have abdominal pain, vomiting, diarrhea and other diseases.

[0003] At present, there are few alternatives to control Vibrio parahaemolyticus in aquaculture, including some disinfectants and a few antibiotics permitted by law. Using antibiotics is a common practice for treating diseases, but overuse of antibiotics may lead to the emergence of antibiotic-resistant bacteria, posing a huge threat to food safety by resistant bacteria or even multi-drug resistant "superbugs". The research on phages has once again attracted the attention of scientific researchers in various countries. However, bacteria can also develop resistance to phages, which limits the application of phages. Therefore, some scientific researchers have focused on the endolysins produced during the process of virulent phages lysing host cells. The bactericidal property with endolysin as the main active substance has many advantages such as being easily and directionally modified by gene editing and not being neutralized by antibodies. It 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 characteristics of food, having the potential to be applied in the food industry.

[0004] Endolysin is an enzyme used by phages to lyse the host cell wall in the final stage of the lysis cycle. Phages containing double-stranded DNA can lyse the peptidoglycan layer by producing endolysin in the final stage of the lysis cycle. This mechanism represents a promising enzyme-based antibacterial effect. The bactericide with endolysin as the main active substance has the following advantages in controlling bacterial pathogens in food: (i) rapidly reducing target bacteria; (ii) expanding the antibacterial spectrum compared with phages; (iii) reducing the resistance rate; and (iv) improving the safety of food applications without producing any toxins. Many studies related to endolysin have shown that endolysin can kill Escherichia coli O157:H7, Listeria monocytogenes, Campylobacter jejuni, Salmonella spp. and Pseudomonas spp. So far, a representative phage lysin recombinant protein P128 is in the late stage of preclinical drug development for the local prevention and treatment of staphylococcal skin infections. However, there is still no commercially available endolysin bactericide for Vibrio.

[0005] In summary, exploring a new endolysin with significant antibacterial effect for the prevention and control of Vibrio parahaemolyticus has great value. Summary of the Invention

[0006] Based on the technical problems existing in the background art, the present invention provides a Vibrio parahaemolyticus phage endolysin LysC75 and its application, which can effectively inhibit the growth of Vibrio parahaemolyticus, have strong specificity for Vibrio parahaemolyticus and high lysis efficiency, and can be used for the prevention and control of Vibrio parahaemolyticus.

[0007] A Vibrio parahaemolyticus phage endolysin LysC75 provided by the present invention, the amino acid sequence of the endolysin LysC75 is shown in SEQ ID NO.2.

[0008] A DNA provided by the present invention, which contains the gene encoding the above-mentioned Vibrio parahaemolyticus phage endolysin LysC75, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0009] An expression vector provided by the present invention contains the above DNA.

[0010] A microorganism provided by the present invention is transformed by using the above expression vector.

[0011] The application of the above-mentioned Vibrio parahaemolyticus phage endolysin LysC75 in a bactericide.

[0012] An enzyme preparation provided by the present invention contains the above-mentioned Vibrio parahaemolyticus phage endolysin LysC75.

[0013] The beneficial technical effects of the present invention: The present invention provides a bactericide with Vibrio parahaemolyticus endolysin as the main active substance. This bactericide 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. The bactericidal effect shows a dose dependence. The Vibrio parahaemolyticus concentration can be reduced from 10 8 CFU / mL to 10 5 CFU / mL by the bactericide with a concentration of 1mg / mL within 120 min, and the bactericide with a concentration of 0.3mg / mL can reduce the bacterial concentration from 10 8 CFU / mL to 10 7 CFU / mL. The bactericidal effect of the bactericide with a concentration of 1mg / mL on Vibrio parahaemolyticus with a concentration of ≤10 3 CFU / mL is 100%. The bactericidal rate for strains with a concentration of 10 6 -10 4 CFU / mL can reach more than 90%, and the bactericidal rate for strains with a concentration of 10 9 -10 6 CFU / mL can reach more than 90%.

[0014] A new virulent phage against multi-drug resistant bacteria was screened and isolated from the sewage of a sewage treatment plant. Through the study of its properties, it was found that the phage has a wide host spectrum, good temperature and pH stability, and good antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the plaque map of phage C75 proposed by the present invention.

[0016] Figure 2 It is the electron microscope image of phage C75 proposed by the present invention.

[0017] Figure 3 It is the schematic diagram of the multiplicity of infection of phage C75 proposed by the present invention.

[0018] Figure 4 It is the schematic diagram of the one-step growth curve of phage C75 proposed by the present invention.

[0019] Figure 5 It is the VIRIDIC heat map of phage C75 proposed by the present invention.

[0020] Figure 6 It is the heat map of genomic comparison of different phages by Easyfig proposed by the present invention.

[0021] Figure 7 It is the phylogenetic tree of endolysin LysC75 proposed by the present invention.

[0022] Figure 8 The recombinant plasmid map proposed by the present invention.

[0023] Figure 9 It is the protein electrophoresis map of the purified recombinant expressed phage endolysin LysC75 proposed by the present invention.

[0024] Figure 10 It is the determination map of the antibacterial activity of phage endolysin LysC75 proposed by the present invention.

[0025] Figure 11 It is the bactericidal effect map of endolysin bactericide at 0 min proposed by the present invention.

[0026] Figure 12 It is the bactericidal effect map of endolysin bactericide at 30 min proposed by the present invention.

[0027] Figure 13 It is the bactericidal effect map of endolysin bactericide at 60 min proposed by the present invention.

[0028] Figure 14 It is the bactericidal effect map of endolysin bactericide at 90 min proposed by the present invention.

[0029] Figure 15 The bactericidal effect diagram of the endolysin bactericide proposed by the present invention after 120 minutes

[0030] Figure 16 The bactericidal effect diagrams of the endolysin bactericide proposed by the present invention at different times and different concentrations

[0031] Figure 17 The bactericidal effect diagrams of the endolysin bactericide proposed by the present invention against bacteria at different concentrations

[0032] Figure 18 The temperature stability of the endolysin bactericide proposed by the present invention

[0033] Figure 19 The pH stability of the endolysin bactericide proposed by the present invention Detailed implementation manners

[0034] The present invention will be further explained below in conjunction with specific embodiments

[0035] The phage C75 in the present invention is preserved in the China Center for Type Culture Collection, address: Preservation Center of Wuhan University, preservation number CCTCC M 2025326, preservation date: February 28, 2025, classification name: Vibrio parahaemolyticus phage vB_VpaP_XY75

[0036] The strains in the host spectrum of phage C75 in Table 1 are from the preservation of this laboratory

[0037] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels without special instructions Example 1

[0038] Isolation and purification of phage Water sample treatment The water sample was collected from the sewage of a sewage treatment plant. The water sample was centrifuged at 10000g for 10 minutes to remove large solid particles and some bacteria. The supernatant was taken. The supernatant was vacuum filtered through a 0.45mm and water-based filter membrane into a clean and sterile conical flask. MgSO4 with a final concentration of 50 mM was added, stirred evenly, and left standing for 15 - 20 minutes. The mixed solution was vacuum filtered, the supernatant was discarded, the filter membrane was collected, the filter membrane was cut into pieces and put into a beaker containing an appropriate amount of eluent, and ultrasonicated for 4 - 5 minutes to elute the phage particles from the filter membrane. The mixed solution was filtered through a 0.45mm filter head into another dry and sterile conical flask. This liquid is the phage mixed solution and is stored at 4°C for later use

[0039] Inoculate Vibrio parahaemolyticus under aseptic conditions into 5 mL of TSB medium and culture overnight. Inoculate the activated bacterial solution into a new 5 mL of TSB medium at an inoculation amount of 1% and culture until the logarithmic phase. Take 500 mL of phage mixture, 500 mL of double-strength TSB medium (containing CaCl2 at a final concentration of 2 mM), and 50 mL of logarithmic-phase bacterial solution, mix them evenly, and culture with shaking at 37 °C for 8 h. Centrifuge the culture solution at 4000 g for 15 - 20 min, and filter it through a 0.45 mm filter head into a sterile centrifuge tube to obtain the phage lysate, which is stored at 4 °C for later use.

[0040] Phage purification Pour 1.5% TSA (tryptic soy agar medium) onto a clean and sterile petri dish. Take 100 mL of bacterial solution cultured to the logarithmic phase and 100 mL of phage lysate, add them to 5 mL of 0.7% TSB-Ca soft agar, mix evenly, and spread it on the surface of the dry 1.5% TSB agar. Incubate statically at 37 °C for 8 h to obtain clearly visible plaques. Use a sterile micropipette tip to pick a single transparent plaque and dissolve it in an appropriate amount of SM buffer, mix well, centrifuge at 4000 g for 15 - 20 min, collect the supernatant, and filter the supernatant through a 0.45 mm filter head to obtain the phage purified solution. Repeat the purification multiple times until the plaque morphology is single and transparent ( Figure 1 )

[0041] Phage proliferation and concentration Take 1 mL of TSB-Ca broth culture medium in a sterile centrifuge tube, add 20 mL of bacterial solution cultured to the logarithmic phase and 20 mL of phage purified solution, and culture at 37 °C and 200 rpm for 8 h. Centrifuge at 4000 g for 15 - 20 min, filter through a 0.45 mm filter head into a new sterile centrifuge tube to obtain the phage proliferation solution. Add NaCl at a final concentration of 0.5 M and 10% polyethylene glycol 8000 to the proliferation solution, and let the phage particles sediment overnight at 4 °C. Centrifuge at 12000 g for 10 min to remove the supernatant, collect the precipitate, and redissolve the precipitate in SM buffer to obtain the phage concentrated solution. Store at 4 °C for later use. (For long-term storage, add glycerol at a final concentration of 30% and store at -20 °C.) Measure its titer by the double-layer agar plate method. Example 2

[0042] Observation of phage morphology Drop the purified phage suspension on a copper grid, after 3 - 5 min, suck it off with filter paper, drop a drop of PTA, stain for 2 - 3 min, then suck off the excess liquid, dry it in the air, and then observe with a field emission transmission electron microscope. Phage C75 belongs to the Myoviridae family, with a head of about 40 mm and a tail of about 62 mm ( Figure 2 ) Example 3

[0043] Determination of the host spectrum of phage C75 Spread 1.5% TSB agar evenly onto a dry and sterile petri dish and wait for it to dry. Add 100 mL of the bacterial liquid cultured to the logarithmic phase into 5 mL of 0.4% TSA, mix well, spread it evenly onto the dried plate, and let it dry naturally to solidify the soft agar. Add 2 mL of the phage culture solution to the soft agar by spotting method, let it dry naturally, and then culture it at 37 °C for 4 - 6 h. The lysis effect is divided into clear spotting area (+) and no plaque in the spotting area (-). The results are shown in Table 1. The host spectrum of C75 was determined using 27 strains of bacteria. The results showed that the bactericidal rate of phage C75 was 44%, and it had no infectivity to non-Vibrio parahaemolyticus strains, indicating its good specificity.

[0044] Table 1 Results of the determination of the host spectrum of phage C75 Serial number Strain name Bacterial species Lysis effect 1 ATCC17802 <![CDATA - > 2 GD91 + 3 GD103 + 4 GD71 + 5 GD78 <![CDATA - > 6 GD81 <![CDATA - > 7 GD62 + 8 GD75 + 9 GD74 + 10 GD89 + 11 GD80 + 12 GD83 + 13 GD73 <![CDATA - > 14 99-1 <![CDATA - > 15 66-1 <![CDATA - > 16 124-1 <![CDATA - > 17 84-1 + 18 163-1 <![CDATA - > 19 234-2 + 20 234-1 <![CDATA - > 21 84-5 + 22 ATCC 13076 <![CDATA - > 23 ATCC 19111 <![CDATA - > 24 ATCC 51329 <![CDATA - > 25 ATCC 19433 <![CDATA - > 26 ATCC 14028 <![CDATA - > 27 ATCC 49128 <![CDATA - > Example 4

[0045] Determination of the optimal multiplicity of infection of phage Culture the bacteria to the early logarithmic phase to make the bacterial concentration 10 8 cfu / mL. Add the phage liquid and the host bacterial liquid according to the ratios of multiplicity of infection of 100:1, 10:1, 1:1, 1:10, 1:100, 1:1000, and 1:10000. Incubate at 37 °C and 200 rpm for 4 h, centrifuge at 12000 rpm / min for 10 - 15 min, filter through a 0.45 µm filter head, and measure the titer by the double-layer agar plate method. The multiplicity of infection that produces the highest titer is the optimal infection.

[0046] As Figure 3 shown, when MOI = 1:100, the titer is the highest, which is 8.1×10 10 pfu / mL, that is, the optimal MOI of phage C75 is 1:100. Example 5

[0047] Determination of the one-step growth curve of phage Culture the bacteria to the early logarithmic phase to make the bacterial concentration 10 8 cfu / mL. Add the host bacteria and the phage culture solution according to the optimal multiplicity of infection of 1:100 and culture with shaking at 37 °C. Sampling is carried out every 5 min for the first 20 min and every 10 min thereafter at 0 min. Centrifuge at 12000 rpm / min for 30 s, filter through a 0.45 mm filter head, and measure the phage titer at each time point. Draw a one-step growth curve with the infection time as the abscissa and the phage titer as the ordinate.

[0048] As Figure 4As shown in the figure, the adsorption period of phage C75 is 0 - 5 min, the lysis period is 10 - 20 min, and the stationary period is 30 - 120 min. Example 6

[0049] Phage Genome Sequencing and Analysis Add DNase I and RNase A to the 500 mL phage concentrate in Example 1, incubate at 37 °C for 60 min (invert every 30 min). Add 10% SDS, proteinase K, and EDTA, incubate at 65 °C for 60 min (invert 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 upper aqueous phase 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 upper aqueous phase to a new centrifuge tube. Extract with an equal volume of chloroform, vortex for 30 s, centrifuge at 12000 g for 5 min, transfer the upper aqueous phase to a new centrifuge tube, and repeat this operation twice. Add an equal volume of isopropanol, mix well, place at -20 °C for 30 min, centrifuge at 12000 g at 4 °C for 20 min, and collect the precipitate. Wash the precipitate with 200 mL of 70% ethanol, centrifuge at 12000 g for 5 min, discard the supernatant, retain the precipitate, and repeat twice. Dry the DNA precipitate at room temperature, add 20 mL of sterile water preheated at 65 °C, and store at -20 °C for later use.

[0050] From the Illumina sequencing results, it can be seen that phage C75 is a linear, dsDNA phage. The complete genome sequence of phage C75 is 44860 bp long, with a GC content of 48.68%, and contains 50 coding sequences (CDS). Compared with the virulence database and the antibiotic resistance gene database, phage C75 does not contain virulence genes and antibiotic genes, indicating that it can be safely used to control Vibrio parahaemolyticus. Figure 5 VIRIDIC heatmap of phage C75. The similarity between phage C75 and Vibrio phage vB_VpaP_MGD1 is 100% (coverage rate 96.03%); the similarity between phage C75 and Vibrio phage vB_VpaP_1701 is 98% (coverage rate 95.28%).

[0051] Figure 6This is a heatmap of genomic comparison of different phages by Easyfig. Phage C75 is predicted to contain 50 ORFs and no tRNAs. Among them, 26 ORFs are annotated as functional proteins, and the rest are annotated as hypothetical proteins, with no tRNAs. The functional proteins of the phage are divided into five modules: DNA metabolism module (DNA metabolism), lysis module (Lysis), packaging module (Packaging), structure module (Structure), and other functional modules (Additional function). Example 7

[0052] Endolysin protein expression After sequencing its whole genome and uploading the results to the ncbi database, the original coding nucleotide sequence of phage endolysin LysC75 obtained by blast alignment is shown as SEQ ID NO 1: ATGCTAATTAAAGACACTGCGAATTTCAAGATTACGAAGTTCGCGTGTCAGCATTGTGGGGCTTTGAAACTAGACCTAGCCCTGCTTATGCTGGTACAAATGCTACGGGAGCACTTCGGAGAACCGCTAAAGGTTGAATCCGGTTATCGCTGCCCTGTACACAATAAAGCCGTAGGCGGTGCTGAGGACTCTCGTCACTTACATGGTGATGCAGTGGACTTGCACTTGCTGAACAAAGACCGGGGGAACTTCCAGAAGCTCCAGAAGCTGTACGACACGGCTTTAGCTCTGAACCCTAACGGTGGCGTCGGTCTGTACGACTGGGGTGTACACGTTGATACACGCGGTGAGAAAGCCCGTTGGGATTACCGCTCTGATAAATACAAAGAAGTAATGGGGAAAATGGATGTCTGA The amino acid sequence corresponding to phage endolysin LysC75 is shown as SEQ ID NO.2: MLIKDTANFKITKFACQHCGALKLDLALLMLVQMLREHFGEPLKVESGYRCPVHNKAVGGAEDSRHLHGDAVDLHLLNKDRGNFQKLQKLYDTALALNPNGGVGLYDWGVHVDTRGEKARWDYRSDKYKEVMGKMDV As Figure 7As shown in the figure, by analyzing the endolysin phylogenetic tree, it was found that the phage endolysin LysC75 had a high homology with the lyase in vB_VpaP_MGD1.

[0053] According to the method of artificial gene synthesis, it was added to the EcoR I and Xho I restriction sites of the pGEX-4T-1 plasmid to obtain the recombinant plasmid pGEX-4T-1-LysC75, which was then introduced into Escherichia coli BL21 competent cells to obtain a positive clone strain containing the pGEX-4T-1-LysC75 plasmid. The specific procedure was as follows: Using the phage endolysin genome as a template, a gene of approximately 500 bp was successfully amplified with primers. The size of the PCR product was consistent with the expected target fragment (414 bp). The plasmid pGEX-4T-1 and the PCR product purified by gel recovery were digested with the restriction enzymes XholI and EcoRI. After verifying the correct target band by 1.5% agarose gel electrophoresis, the gel was recovered to obtain the plasmid and the target gene with the same sticky ends. The above-obtained vector and target gene with sticky ends were ligated with T4 ligase, and then the ligated recombinant plasmid was transformed into the competent cell BL21. The sequencing results showed that the nucleotide sequence was correct, indicating the successful construction of the recombinant plasmid. The recombinant plasmid map is as Figure 8 shown.

[0054] The primers were as follows: Forward primer (SEQ ID NO 3): CCCTCGAGTCAGACATCCATTTTCCCC Reverse primer (SEQ ID NO 4): CGGAATTCATGCTAATTAAAGACACTGCGA The positive clone strain was cultured and induced for protein expression to obtain the Vibrio parahaemolyticus phage endolysin LysC75, which was then enriched and concentrated. IPTG was used to induce the expression of the vector fusion protein, specifically as follows: (1) The recombinant bacteria were cultured overnight and inoculated into LB broth medium containing Amp (final concentration 50 μg / mL) at a ratio of 1%. The recombinant bacteria were cultured at 37 °C and 200 rpm until OD 600 = 0.6 - 0.8; then isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM was added to the cultured recombinant bacteria, and induction was carried out at 16 °C and 200 rpm for 12 - 16 h.

[0055] (2) Centrifuge the bacterial solution at 4°C and 10,000 g for 10 min, discard the supernatant, and resuspend the bacterial cells in cell lysis buffer. Add phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM before sonication to prevent protein denaturation. Sonicate the bacterial cells (power: 70 W, work for 4 s, stop for 8 s, for a total of 10 min). Centrifuge the lysate at 4°C and 12,000 g for 30 min, collect the supernatant, and the supernatant is the crude protein.

[0056] Purify the crude protein using the steps in the instruction manual of Mag-Beads GST fusion protein purification magnetic beads. Prepare an SDS-PAGE gel (5% stacking gel, 12% separating gel); after mixing the flow-through sample, wash sample, and elution sample of the purified protein above with protein loading buffer, boil at 100°C for 3 - 5 min, centrifuge at 12,000 g for 2 - 5 min, and load the supernatant for electrophoresis. After electrophoresis, stain with Coomassie Brilliant Blue and then decolorize three times with distilled water.

[0057] As Figure 9 shown in the SDS-PAGE pattern, lanes 1 - 7 indicate that impurities are removed after 4 washes. The 8th lane is the eluate of the endolysin purified by magnetic beads. The size of the target band is 42 kd and only one band appears, proving the successful expression and purification of the endolysin.

[0058] Add the purified protein solution into a dialysis bag, and the ratio of the protein amount to the dialysis solution is 1:10. Place it in the dialysis solution and in a 4°C refrigerator, and stir magnetically for 12 h. Centrifuge the dialyzed protein at 12,000 rpm and 4°C for 20 min, remove the precipitate, and load the supernatant into a 30,000 KD ultrafiltration tube and ultrafiltrate and concentrate it at 4°C and 3,000 g. Example 8

[0059] Culture the GD83 bacterial solution to 10 8 CFU / mL, centrifuge at 8,000 g for 10 min, discard the supernatant, add sterile saline buffer to wash the precipitate, repeat the washing twice, add an appropriate amount of saline buffer to resuspend the bacterial cells, and make the absorbance OD 600 of the resuspended bacterial cells = 1. Design 6 treatment groups based on the resuspension: ① no addition; ② add 5 mM EDTA; ③ add 1 mg / mL endolysin solution; ④ add 1 mg / mL endolysin solution and 5 mM EDTA permeabilizer; ⑤ add 0.1 mg / mL endolysin solution; ⑥ add 0.1 mg / mL endolysin solution and 5 mM EDTA permeabilizer. Incubate in a 37°C environment for 5 min, 30 min, 60 min, 90 min, 120 min, and use a UV spectrophotometer to measure the OD 600 of the mixed solution to reflect the bacterial concentration.

[0060] The results are as follows Figure 10 shown. The difference between using EDTA alone and the control group is small; using lysin alone cannot effectively kill Vibrio parahaemolyticus, and the bactericide has a significantly better effect than using EDTA alone and using lysin alone. The bactericidal effect of lysin appears within 5 min, and the bactericidal efficiency of bacteria is the highest from 5 to 30 min. The bactericidal effect shows concentration dependence. Adding lysin with a final concentration of 1 mg / mL has a significantly better effect than that with a final concentration of 0.1 mg / mL, indicating that the higher the concentration of lysin in the preparation, the better the bactericidal effect.

[0061] Cultivate the GD80 bacterial solution to 10 8 CFU / mL, centrifuge at 8000 g for 10 min to precipitate the bacteria at the bottom of the centrifuge tube, discard the supernatant, wash the precipitate twice, and add an equal volume of sterile normal saline to resuspend the bacteria to make the bacterial concentration 10 8 CFU / mL. Add bactericides with different concentrations to the bacterial solution respectively, and use the addition of sterile normal saline buffer as the negative control. Incubate the mixture at 37 °C for 0 min, 30 min, 60 min, 90 min, and 120 min respectively.

[0062] Figures 11 - 15 They are the bactericidal effect diagrams at 0 min, 30 min, 60 min, 90 min, and 120 min under the concentration gradient conditions of 1 - 0.1 mg / mL respectively. Figure 16 For Figures 11 - 15 the corresponding bactericidal effect quantity of the experiment. The effect of the bactericide shows concentration dependence, and the bactericidal efficiency of the bactericide is the highest within 30 min. The effect of the bactericide shows concentration dependence, and the bactericidal efficiency of the bactericide is the highest within 30 min. Under the condition of 1 mg / mL bactericide, the concentration of Vibrio parahaemolyticus can be reduced from 10 8 CFU / mL to 10 6 CFU / mL in 30 min, and the best bactericidal effect is achieved in 2 h. Finally, the concentration of Vibrio parahaemolyticus is reduced from 10 8 CFU / mL to 10 5 CFU / mL. The higher the concentration of the bactericide within the concentration range of 0.1 - 1 mg / mL lysin, the better the effect. The lowest effective concentration of the bactericide is 0.3 mg / mL. The concentration of Vibrio parahaemolyticus can be reduced from 10 8 CFU / mL to 10 7 CFU / mL in 120 min.

[0063] Cultivate the GD80 bacterial solution to 10 8 CFU / mL, centrifuge at 8000 g for 10 min to precipitate the bacteria at the bottom of the centrifuge tube, discard the supernatant, wash the precipitate twice, and make the concentration of the bacteria at 10 9 、10 8 、107 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 Add lysozyme and EDTA to the bacterial suspension at 10 CFU / mL respectively, and use the one with PBS buffer added as the negative control. Incubate the mixture at 37 °C for 2 h respectively, and count using the PCA plate counting method.

[0064] Figure 17 Figure shows the bactericidal effect of the bactericide on bacterial suspensions at different concentrations. When the bacterial concentration is 10 CFU / mL and below, the clearance rate of the bactericide reaches 100%, and it can effectively deal with Vibrio parahaemolyticus infection in the environment. Even under high-concentration bacterial conditions (bacterial concentration is 10 - 10 CFU / mL), the bactericidal rate of the bactericide can still reach more than 90%. 3 CFU / mL and below, the clearance rate of the bactericide reaches 100%, and it can effectively deal with Vibrio parahaemolyticus infection in the environment. Even under high-concentration bacterial conditions (bacterial concentration is 10 9 -10 4 CFU / mL), the bactericidal rate of the bactericide can still reach more than 90%. Example 9

[0065] Determine the stability at different temperatures and pH values. Culture the GD80 bacterial suspension to 108 CFU / mL, centrifuge at 8000 g for 10 min to precipitate the bacteria at the bottom of the centrifuge tube, discard the supernatant, and wash the precipitate twice. Take 100 mL of LysC75 with a concentration of 10 mg / mL in a 1.5 mL centrifuge tube, and incubate at 4 °C, 10 °C, 20 °C, 30 °C, 37 °C, 50 °C, 60 °C, 70 °C for 1 h respectively. After the reaction, take it out and cool it on ice, add it to the resuspended GD80 bacterial suspension, and incubate at 37 °C for 2 h, and count the GD80 bacterial concentration by plate counting. Take 100 mL of LysC75 with a concentration of 10 mg / mL in a 1.5 mL centrifuge tube, add different pH values (50 mM citrate buffer pH 3 - 6, 50 mM, and 50 mM tris-HCl buffer pH 7 - 9) respectively, incubate at 37 °C for 2 h, and count the GD80 bacterial concentration by plate counting.

[0066] The results are as follows Figure 18 , Figure 19 shown. After the bactericide acts at 4 °C - 37 °C for 1 h, the bactericidal effect hardly changes. After the bactericide is treated at 50 °C for 1 h, the bactericidal rate decreases by 50% and the titer decreases. After the bactericide acts at 60 °C and 70 °C for 1 h, the bactericide is completely inactivated. In an environment with a pH of 4 - 9, the bactericidal effect of the bactericide hardly changes. When the pH is below 5, the bactericide is completely inactivated.

[0067] Although embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application. The scope of the present application is defined by the appended claims and their equivalents, and all of them should be included within the protection scope of the present application.

Claims

1. A Vibrio parahaemolyticus bacteriophage endolysin LysC75, characterized in that, The amino acid sequence of endolysin LysC75 is shown in SEQ ID NO.

2.

2. A DNA, characterized in that, It contains the gene encoding the endolysin LysC75 of Vibrio parahaemolyticus phage described in claim 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. An expression vector, characterized in that, It contains the DNA described in claim 2.

4. A microorganism, characterized in that, Transformation is carried out using the expression vector described in claim 3.

5. A fungicide, characterized in that, It contains the endolysin LysC75 of Vibrio parahaemolyticus phage described in claim 1.

6. An enzyme preparation, characterized in that, It contains the endolysin LysC75 of Vibrio parahaemolyticus phage described in claim 1.

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