Application of vibrio parahaemolyticus lyase LysV569 in preparation of medicine for splitting vibrio parahaemolyticus

Abstract

The invention provides application of vibrio parahaemolyticus lyase LysV569 in preparation of a medicine for splitting vibrio parahaemolyticus. The amino acid sequence of the vibrio parahaemolyticus lyase LysV569 is shown in SEQ ID NO. 1, and the gene sequence of the vibrio parahaemolyticus lyase LysV569 is shown in SEQ ID NO. 2. The lyase can independently penetrate through the outer membrane of gram-negative bacteria without an outer membrane permeabilizing agent, so that the defect that the lyase depends on a chemical adjuvant in the prior art is overcome; under the condition of no permeabilizing agent, the enzyme shows ultrahigh cracking efficiency (the sterilization rate is greater than 99.9% within 1 hour under the concentration of 100 [mu] g / mL, and the CFU is reduced by 4-log) and thermal stability (the activity is maintained to be greater than 99% after 30 minutes of high-temperature treatment at 85 DEG C), and is suitable for scenes of aquaculture pathogen prevention and control, food industry high-temperature sterilization and drug resistance delay by combination of antibiotics. An efficient, environment-friendly and operation-simplified solution is provided for preventing and controlling the food-borne pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to the application of a Vibrio parahaemolyticus lysin LysV569 in the preparation of drugs for lysing Vibrio parahaemolyticus. Background Technology

[0002] Vibrio parahaemolyticus, a member of the genus Vibrio in the family Vibriocecaceae, is a Gram-negative, halophilic, straight or curved rod-shaped facultative anaerobe naturally found in marine environments. This bacterium is a human pathogen, naturally present in the marine environment, and frequently isolated from various seafood, including cod, sardines, mackerel, halibut, clams, octopus, shrimp, crab, lobster, crayfish, scallops, and oysters. Ingestion of raw or undercooked seafood, especially shellfish, contaminated with Vibrio parahaemolyticus can cause acute gastroenteritis, characterized by diarrhea, headache, vomiting, nausea, abdominal cramps, and low-grade fever. It is a significant foodborne pathogen worldwide. This bacterium not only causes human pathogens but also severely impacts aquaculture, resulting in substantial economic losses. In aquaculture, infectious disease control is typically achieved through antibiotic administration. However, due to the extensive and inappropriate use of antibiotics in aquaculture and clinical systems, Vibrio parahaemolyticus is developing increasing resistance to antibiotics, leading to a decline in the effectiveness of antibiotic-based treatments. Therefore, there is an urgent need to develop effective biocontrol agents to combat Vibrio parahaemolyticus contamination.

[0003] Bacteriophages are bacterial viruses that can attack and kill target bacteria within minutes of infection, and are considered potential antibacterial agents due to their excellent specificity and ease of isolation. The application of bacteriophages as antibacterial agents, known as phage therapy, was widely practiced in the 1920s. Phage lyases are peptidoglycan hydrolases encoded by bacteriophages that enzymatically degrade the peptidoglycan layer of host bacteria "from the inside" at the end of the phage lysis cycle. Compared to live phages, phage lyases offer a wider lysis spectrum, accelerate antibacterial efficacy, reduce the likelihood of host resistance development, and have negligible impact on beneficial bacteria and the microbial ecosystem. However, few lyase proteins have been identified from Vibrio-specific phages. In the cell structure of Gram-negative bacteria, an outer membrane structure exists outside the peptidoglycan layer, which prevents phage lyases from entering and lysing the bacteria. Currently, there is a lack of effective vibrio lyases that can permeate the outer membrane. Therefore, it is necessary to develop novel phage lyases with highly efficient lysis activity against Vibrio parahaemolyticus. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Vibrio parahaemolyticus lysin LysV569 in the preparation of a drug for lysing Vibrio parahaemolyticus, wherein the Vibrio parahaemolyticus lysin LysV569 is described.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect of the invention, a Vibrio parahaemolyticus lysin LysV569 is provided, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0006] amino acid sequence: MASIRISGSVGLGGKNVDADIRTVQRSINQLLGSLKGVKELKVDGKLGSRPENSKTVAAIKAFQKNLVGMARPDGRIDVNGRSHRKLNEYLKRTPEIAVAYTLPLVGSRDALTDLDYSKVAETLGCEVAAIKAVAEVESRGDAYFSNGKP KILFEAHIFSRLTSRAYDNSHPSISSRRWNRSLYVGGISEYVRLNKAIELNSNAAIRSASWGRFQIMGFNFKLAGHVTAESFVKAVFESEKKQLEAFVTFIQKSGLGEHIRYKNWAAFARGYNGSEYQKNQYDVKLEKAYKKYASIKNAA; In a second aspect of the invention, a nucleic acid molecule of Vibrio parahaemolyticus lysin LysV569 is provided, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0007] Gene sequence: ; In a third aspect of the invention, an expression vector for Vibrio parahaemolyticus lysin LysV569 is provided, the expression vector comprising encoding the nucleic acid molecule.

[0008] Furthermore, the expression vector includes one of a prokaryotic expression vector and a viral vector.

[0009] In a fourth aspect of the present invention, a method for preparing a Vibrio parahaemolyticus lysin LysV569 expression vector is provided, the method comprising: Obtain the target gene fragment of the nucleic acid molecule of Vibrio parahaemolyticus lysin LysV569 as shown in SEQ ID NO: 2; The target gene fragment and the pET28a expression vector were digested with NcoI and XhoI, and then ligated to obtain the expression vector pET28a-LysV569.

[0010] In a fifth aspect of the invention, a recombinant bacterium or engineered cell line comprising the expression vector is provided.

[0011] In a sixth aspect of the invention, the use of the Vibrio parahaemolyticus lysin LysV569, the nucleic acid molecule, the expression vector, the recombinant bacteria, or the engineered cell line described herein in the preparation of a medicament for lysing Vibrio parahaemolyticus is provided.

[0012] In a seventh aspect of the invention, a medicament for lysing Vibrio parahaemolyticus is provided, the medicament comprising the Vibrio parahaemolyticus lysin LysV569, or the recombinant expression vector expressing the Vibrio parahaemolyticus lysin LysV569, or the recombinant bacteria or engineered cell line containing the expression vector of the Vibrio parahaemolyticus lysin LysV569.

[0013] In an eighth aspect of the invention, a method is provided for lysing a test sample containing Vibrio parahaemolyticus using the Vibrio parahaemolytic enzyme LysV569, the method comprising: The test sample containing Vibrio parahaemolyticus was cultured to the logarithmic or stationary phase, and the precipitate was collected by low-temperature centrifugation to obtain the bacterial solution. The Vibrio parahaemolyticus lysin LysV569 was mixed with the bacterial culture and incubated to obtain a sample after Vibrio parahaemolyticus lysis. Further, the concentration of the Vibrio parahaemolyticus lysin LysV569 used was 25-100 μg / mL.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention provides the application of a Vibrio parahaemolyticus lysin LysV569 in the preparation of drugs for lysing Vibrio parahaemolyticus. This invention screened out a Vibrio parahaemolyticus lysin LysV569, and its breakthrough is verified experimentally as follows: (1) No outer membrane permeation agent required: It independently penetrates the outer membrane of Gram-negative bacteria. The lysin LysV569 has a good lysing effect on Vibrio parahaemolyticus and does not require treatment with an outer membrane permeation agent. The activity of LysV569 is dose-dependent. At a concentration of 25 μg / mL, it can kill more than 99% of bacteria within 1 hour. At a concentration of 100 μg / mL, it can reduce the CFU count by about 4 logs, with a bactericidal rate of more than 99.9%. This solves the defect of existing technologies that rely on chemical permeation agents (existing bacteriophage lysins require outer membrane permeation agents such as Triton X-100 and EDTA to penetrate the outer membrane when acting on Gram-negative bacteria). (2) Ultra-high lysis efficiency: Sterilization rate > 99.9% within 1 hour at a concentration of 100 μg / mL (see Example 2). Figure 2 ); (3) Excellent thermal stability: After treatment at 85°C for 30 minutes, it still maintains >99% activity, see Example 3.4. In the lyase activity test, LysV569 can maintain greater than 99% bactericidal activity at all test temperatures, even at high temperatures such as 65°C, 75°C and 85°C, indicating that it has high thermal stability and a wide applicable temperature range. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 : SDS-PAGE image of LysV569 purification.

[0017] Figure 2 Dose-dependent bactericidal curve.

[0018] Figure 3 Time-sterilization rate relationship (30 minutes > 99%).

[0019] Figure 4 The inhibitory effect of NaCl concentration on activity.

[0020] Figure 5 pH adaptability verification (pH 6-7 is optimal).

[0021] Figure 6 Thermal stability verification (activity maintained at 85℃).

[0022] Figure 7 Serum concentration inhibits activity. Detailed Implementation

[0023] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0024] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0026] The lysin of this application will be described in detail below with reference to the embodiments and experimental data.

[0027] Example 1: Construction of LysV569 expression vector, engineered bacteria, protein expression and purification 1. Construction of recombinant vectors The target gene fragment was synthesized by Tianjin Shuangyu Biotechnology Co., Ltd. (the synthesized gene sequence is shown in SEQ ID NO.2, and the codons were optimized for expression in E. coli). It was digested with NcoI restriction endonuclease and XhoI restriction endonuclease, and then ligated with the vector pET28a, which had been digested with the same NcoI restriction endonuclease and XhoI restriction endonuclease, to obtain the recombinant expression vector pET28a-LysV569. The recombinant expression vector pET28a-LysV569 was then transformed into E. coli BL21(DE3), and positive clones were selected for sequencing verification.

[0028] 2. Expression and purification of lyase E. coli BL21(DE3) cells with correct sequencing and containing pET28a-LysV569 were cultured in 500 mL LB medium containing 50 μg / mL kanamycin until the OD600 reached 0.4–0.6. Then, they were induced with 0.4 mM isopropyl β-D-thiogalactopyranoside at 16 °C for 14–16 h. Afterward, the cells were collected by centrifugation at 4 °C and 5000 rpm for 15 min, washed once with 20 mM imidazole, and resuspended in 20 mM imidazole. The cells were then lysed using a pressure lysate at 4 °C, centrifuged at 4 °C and 8000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter and subjected to affinity chromatography on a nickel column. The fragments eluted with 250 mM imidazole were collected and placed in 20 mM HEPES buffer (pH 10.5) at 4 °C. 7.4) Dialyze overnight to obtain the lysin LysV569. The SDS-PAGE gel image of the purified LysV569 is shown below. Figure 1 .

[0029] from Figure 1 As can be seen, the lysin LysV569 can be obtained in a soluble and high-purity (>95%) form, and the protein band size is around 33 kDa.

[0030] Example 2: The effect of lysin LysV569 on the in vitro lysis of Vibrio parahaemolyticus ATCC 17802. Vibrio parahaemolyticus ATCC 17802 was cultured to the logarithmic growth phase. After centrifugation at low temperature, the precipitate was collected, washed three times with HEPES buffer containing 0.3 mol / L NaCl, and resuspended. The OD600 was adjusted to 0.5. The lysin LysV569 prepared in Example 1 was mixed with the above bacterial culture to achieve final concentrations of 6.25, 12.5, 25, 50, and 100 μg / ml. An equal volume of the buffer and the above bacterial culture mixture was used as a negative control. After incubation at 37°C for 1 hour, TLC was performed, and the results are shown in the figure. Figure 2 .

[0031] from Figure 2 The results show that the lysin LysV569 has a good lytic effect on Vibrio parahaemolyticus, and its activity is dose-dependent without the need for external membrane permeation agent treatment. At a concentration of 25 μg / mL, it can kill more than 99% of the bacteria within 1 hour, and at a concentration of 100 μg / mL, it can reduce the CFU count by about 4 logs, with a bactericidal rate greater than 99.9%.

[0032] Example 3: Effect of LysV569 lysing activity of Vibrio parahaemolyticus ATCC 17802 under different conditions 1. Effect of time on the activity of lysin LysV569 in lysing Vibrio parahaemolyticus ATCC 17802 Vibrio parahaemolyticus ATCC 17802 was cultured to the logarithmic phase. After centrifugation at low temperature, the precipitate was collected, washed three times with HEPES buffer containing 0.3 mol / L NaCl, and resuspended. The OD600 was adjusted to 0.5. The lysin LysV569 prepared in Example 1 was mixed with the above bacterial culture to achieve a final concentration of 100 μg / ml. An equal volume of buffer and the above bacterial culture mixture was used as a negative control. The mixture was incubated at 37°C, and samples were taken at different time points for TLC counting. The results are shown in [Figure number missing]. Figure 3 .

[0033] from Figure 3 The results show that the lyase LysV569 can kill more than 99% of bacteria within 30 minutes.

[0034] 2. Effects of different NaCl concentrations on the lysing activity of LysV569 on Vibrio parahaemolyticus ATCC 17802 Vibrio parahaemolyticus ATCC 17802 was cultured to the logarithmic phase. After centrifugation at low temperature, the precipitate was collected, washed three times with HEPES buffer containing 0.3 mol / L NaCl, and resuspended. The OD600 was adjusted to 0.5. The lysin LysV569 prepared in Example 1 was mixed with the above bacterial culture to a final concentration of 100 μg / ml. NaCl was added to final concentrations of 0.15, 0.2, 0.4, 0.6, 0.8, and 1 mol / L, respectively. An equal volume of buffer and the above bacterial culture mixture was used as a negative control. The mixture was incubated at 37°C for 1 h, and TLC was performed. The results are shown in [Figure number missing]. Figure 4 .

[0035] from Figure 4 The results show that as the concentration of NaCl increases, the lytic activity of the lysin LysV569 on Vibrio parahaemolyticus ATCC 17802 decreases, indicating that NaCl has a significant inhibitory effect on the lysis of Vibrio parahaemolyticus ATCC 17802 by the lysin LysV569.

[0036] 3. Effects of different pH values ​​on the lysing activity of LysV569 on Vibrio parahaemolyticus ATCC 17802 Prepare a series of pH gradients using BR buffer solution: pH=6, pH=7, pH=8, and pH=9. Culture *Vibrio parahaemolyticus* ATCC17802 in the logarithmic growth phase. After centrifugation at low temperature, collect the precipitate, wash three times with HEPES buffer containing 0.3 mol / L NaCl, and resuspend in BR buffer solution at each pH value to obtain *Vibrio parahaemolyticus* ATCC 17802 bacterial suspensions at different pH values. Adjust OD600 to 0.5. Mix the lysin LysV569 prepared in Example 1 with the above bacterial suspensions to achieve a final concentration of 100 μg / ml. Use an equal volume of buffer solution and the above bacterial suspension mixture as a negative control. Incubate at 37°C for 1 hour, then perform TLC counting. The results are shown in [Figure 1]. Figure 5 .

[0037] from Figure 5 The results showed that the lysin LysV569 exhibited poor lytic activity against Vibrio parahaemolyticus ATCC 17802 at pH 9. As the solution pH decreased, the lytic activity of LysV569 against Vibrio parahaemolyticus ATCC 17802 increased, with the highest activity observed at pH 6 and 7. These results indicate that LysV569 exhibits higher lytic activity against Vibrio parahaemolyticus ATCC 17802 under neutral and slightly acidic conditions.

[0038] 4. Effect of different temperatures on the lysing activity of LysV569 on Vibrio parahaemolyticus ATCC 17802 Vibrio parahaemolyticus ATCC 17802 was cultured to the logarithmic phase. After collecting the precipitate by low-temperature centrifugation, it was washed three times with HEPES buffer containing 0.3 mol / L NaCl and resuspended, adjusting OD600 to 0.5. Lysin LysV569 prepared in Example 1 was treated at different temperatures for 30 min, and then mixed with the above bacterial culture to achieve a final concentration of 100 μg / ml. An equal volume of buffer and the above bacterial culture was used as a negative control. After incubation at 37°C for 1 h, TLC was performed, and the results are shown in the figure. Figure 6 .

[0039] from Figure 6 The results show that LysV569 maintains greater than 99% bactericidal activity at all test temperatures, even at high temperatures such as 65℃, 75℃, and 85℃, indicating high thermal stability and a wide applicable temperature range. The results also indicate that temperature has almost no effect on the lysis of Vibrio parahaemolyticus ATCC 17802 by the lysin LysV569.

[0040] 5. Effects of different serum concentrations on the activity of LysV569 lysing Vibrio parahaemolyticus ATCC 17802 Vibrio parahaemolyticus ATCC 17802 was cultured to the logarithmic phase. After centrifugation at low temperature, the precipitate was collected, washed three times with HEPES buffer containing 0.3 mol / L NaCl, and resuspended. The OD600 was adjusted to 0.5. The lysin LysV569 prepared in Example 1 was mixed with the above bacterial culture to a final concentration of 100 μg / ml. Human serum was added to final concentrations of 1%, 2%, 4%, 8%, 12.5%, 25%, and 50%, respectively. An equal volume of buffer and the above bacterial culture mixture was used as a negative control. The mixture was incubated at 37°C for 1 hour, and TLC was performed. The results are shown in [Figure number missing]. Figure 7 .

[0041] from Figure 7 The results showed that as the concentration of human serum increased, the lytic activity of LysV569 in lysing Vibrio parahaemolyticus ATCC 17802 decreased, indicating that serum had a significant inhibitory effect on the lysis of Vibrio parahaemolyticus ATCC 17802 by LysV569.

[0042] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0043] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0044] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0045] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of equivalents of the claims, this invention also intends to include them.

Claims

1. A Vibrio parahaemolyticus lysin LysV569, characterized in that, The amino acid sequence of the lysin LysV569 is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the Vibrio parahaemolyticus lysin LysV569, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. A vector for expressing the Vibrio parahaemolyticus lysin LysV569, characterized in that, The expression vector expresses the lysin of claim 1.

4. The expression vector for Vibrio parahaemolyticus lysin LysV569 according to claim 3, characterized in that, The expression vector includes one of the following: prokaryotic expression vector, eukaryotic expression vector, and viral vector.

5. A method for preparing an expression vector for the Vibrio parahaemolyticus lysin LysV569, characterized in that, The method includes: Obtain the target gene fragment of the nucleic acid molecule of Vibrio parahaemolyticus lysin LysV569 as shown in SEQ ID NO: 2; The target gene fragment and the pET28a expression vector were both digested with NcoI and XhoI, and then ligated to obtain the expression vector pET28a-LysV569.

6. A recombinant bacterium or engineered cell line comprising the expression vector according to any one of claims 3-4.

7. The use of the Vibrio parahaemolyticus lysin LysV569 of claim 1, or the nucleic acid molecule of claim 2, or the expression vector of any of claims 3-4, or the expression vector prepared by claim 5, or the recombinant bacteria or engineered cell line of claim 6 in the preparation of a drug for lysing Vibrio parahaemolyticus.

8. A drug for lysing Vibrio parahaemolyticus, characterized in that, The drug comprises the Vibrio parahaemolyticus lysin LysV569 of claim 1, or the recombinant expression vector of the Vibrio parahaemolyticus lysin LysV569 of any one of claims 2-4, or the recombinant bacteria or engineered cell line containing the expression vector of the Vibrio parahaemolyticus lysin LysV569 of claim 5.

9. A method for lysing a test sample containing Vibrio parahaemolyticus using the Vibrio parahaemolytic enzyme LysV569, characterized in that, The method includes: The test sample containing Vibrio parahaemolyticus was cultured to the logarithmic or stationary phase, and the precipitate was collected by low-temperature centrifugation to obtain the bacterial solution. The Vibrio parahaemolyticus lysin LysV569 was mixed with the bacterial solution and incubated to obtain a sample after Vibrio parahaemolyticus was lysed.

10. The method according to claim 9, characterized in that, The concentration of the Vibrio parahaemolyticus lysin LysV569 used is 25-100 μg / mL.

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