Antibacterial protein lp1z_01335 derived from lactobacillus plantarum and application thereof

Abstract

The application discloses an antibacterial protein LP1Z_01335 derived from lactobacillus plantarum and application thereof. The amino acid sequence of the antibacterial protein LP1Z_01335 is shown as SEQ ID NO. 2 or SEQ ID NO. 4, and the nucleotide sequence is shown as SEQ ID NO. 2 or the codon-optimized SEQ ID NO. 3. The antibacterial protein LP1Z_01335 can antagonize staphylococcus aureus, escherichia coli, helicobacter pylori and multidrug-resistant helicobacter pylori. Compared with traditional antibiotic drugs for resisting helicobacter pylori, the application has the advantages of no toxic side effect, no residual risk and green safety on ecological environment. The antibacterial protein preparation has wider pH, high-temperature resistance and enzyme stability, can adapt to more application scenarios, and has great potential in in-vitro antibacterial disinfection, in-vivo drug and other applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antimicrobial protein (LP1Z_01335) derived from Lactobacillus plantarum and its applications. Background Technology

[0002] Helicobacter pylori (Hp) is a Gram-negative bacterium, and its infection is one of the key factors leading to gastrointestinal diseases in humans. Therefore, preventing and treating Hp infection is an effective strategy to prevent gastritis, gastric ulcers, and even gastric cancer, and it is also a research hotspot for gastroenterologists and scholars. Currently, Hp eradication in China mainly uses antibiotics combined with proton pump inhibitors and bismuth preparations. At the same time, antibiotic resistance is increasing year by year, and the overall antibiotic resistance rate of Hp in my country is very high, especially in the southeastern coastal cities where it reaches as high as 95.4%. Therefore, finding a non-antibiotic substance that can antagonize Hp for the treatment of chronic inflammation caused by Hp infection in the medical field is extremely urgent.

[0003] In previous studies, we collected fecal samples from healthy centenarians in Meizhou City, Guangdong Province, China, a world-renowned longevity village, and isolated a strain of *Lactobacillus plantarum* LP1Z. This probiotic exhibits highly effective antagonistic activity against *Helicobacter pylori* (Hp) and even multidrug-resistant *Hp*. Using comparative genomics, we discovered a specific functional gene in this *Lactobacillus plantarum* strain, annotated as a putative cell wall hydrolase protein containing the LysM, PRK13914, and PRK06347 domains. LysM is a widely distributed protein motif whose function is primarily related to the binding of peptidoglycan and chitin, and it is most likely to recognize the N-acetylglucosamine moiety. It was initially found in enzymes that degrade bacterial cell walls, and most LysM-containing bacterial proteins are peptidoglycan hydrolases, but proteins involved in many other biological functions also contain this domain. The PRK13914 domain may be associated with the function of invasion-related endopeptidases. The PRK06347 domain may be associated with the function of 1,4-β-N-acetyl cell wall aminoacyl hydrolases. This hypothetical protein is an extracellular protein, predicted to possess peptidoglycan binding, antibacterial, and 1,4-β-N-acetyl cell wall aminoacyl hydrolase functions. Therefore, it is presumed that this hypothetical protein is an antibacterial protein. Peptidoglycan is present in the cell walls of both Gram-positive and Gram-negative bacteria, so it is presumed that this antibacterial protein has bactericidal effects against both Gram-positive and Gram-negative bacteria. The antibacterial effect of this antibacterial protein is also confirmed in the examples below.

[0004] Currently, the database does not classify this antimicrobial protein functionally, only defining it as a LysM domain-containing protein. Furthermore, there are no reports on its antibacterial, anti-Helicobacter pylori, or even anti-multidrug-resistant Hp properties. LP1Z_01335 is an antimicrobial protein derived from probiotics and has a high safety profile for human use. Therefore, exploring new functions of this protein to efficiently antagonize multidrug-resistant Hp, clarifying its excellent properties of high temperature and low pH tolerance and high enzyme stability, and determining the optimal conditions for culture, induction, and purification through multiple experiments to maximize protein yield and purity are urgent technical problems to be solved by researchers in this field. At the same time, summarizing and generalizing the anti-Hp mechanism, physicochemical properties, and preparation methods of this antimicrobial protein is also particularly important. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide an antimicrobial protein, which is derived from Lactobacillus plantarum and has high activity against multidrug-resistant Helicobacter pylori (Hp).

[0006] The present invention achieves its objective by employing the following technical solutions:

[0007] The first objective of this invention is to provide an antimicrobial protein LP1Z_01335, which is derived from Lactobacillus plantarum and has an amino acid sequence as shown in SEQ ID NO.1, consisting of 352 amino acids; or as shown in SEQ ID NO.4.

[0008] A second objective of this invention is to provide the encoding gene of the aforementioned antimicrobial protein LP1Z_01335.

[0009] Preferably, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.2, or as shown in SEQ ID NO.3 with codon optimization.

[0010] A third object of the present invention is to provide the use of the antimicrobial protein LP1Z_01335 in any of the following:

[0011] a. Use in the preparation of antimicrobial drugs, drugs for the prevention and / or treatment of Helicobacter pylori infection, or drugs for the treatment of gastrointestinal diseases;

[0012] b. Application in the preparation of antibacterial agents or disinfectants.

[0013] Preferably, the antibacterial drug is an antibacterial drug against Staphylococcus aureus, Escherichia coli, or Helicobacter pylori.

[0014] A fourth objective of this invention is to provide a recombinant vector containing the encoding gene of the antimicrobial protein LP1Z_01335.

[0015] Preferably, it contains the nucleotide sequence shown in SEQ ID NO.3.

[0016] A fifth objective of this invention is to provide a recombinant engineered bacterium containing the aforementioned recombinant vector.

[0017] The sixth object of the present invention is to provide a method for preparing the above-mentioned antimicrobial protein LP1Z_01335, characterized in that the above-mentioned encoding gene or recombinant vector is expressed in microorganisms and the antimicrobial protein is prepared.

[0018] Preferably, the microorganism is Escherichia coli.

[0019] A seventh object of the present invention is to provide an enzyme preparation comprising the above-mentioned antimicrobial protein LP1Z_01335 as an active ingredient.

[0020] Preferably, the enzyme preparation is an enzyme preparation that antagonizes Staphylococcus aureus, Escherichia coli, Helicobacter pylori, or multidrug-resistant Helicobacter pylori. This enzyme preparation can not only antagonize common Gram-positive and Gram-negative bacteria such as Staphylococcus aureus and Escherichia coli, but also antagonize Helicobacter pylori (G-) and even multidrug-resistant Helicobacter pylori in acidic environments such as the stomach, and has good enzyme stability.

[0021] The beneficial effects of this invention are as follows:

[0022] The antimicrobial protein of this invention exhibits good pH and high-temperature tolerance and enzyme stability. The antimicrobial protein formulation of this invention retains 88.3% or more of its antibacterial activity within a pH range of 3.0 to 9.0, enabling it to exert better antibacterial effects against Helicobacter pylori, even multidrug-resistant Helicobacter pylori, under extreme environments such as the stomach. Within a temperature range of 37°C to 100°C, it retains 99.6% of its antibacterial activity, making it adaptable to a wider range of industrial production processes. After treatment with pepsin, trypsin, and papain, the antibacterial activity is retained at 60.9%, 88.1%, and 99.9%, respectively, indicating the stability of the antimicrobial protein of this invention.

[0023] With a wider pH range, higher temperature tolerance, and enzyme stability, LP1Z_01335 possesses superior enzymatic properties compared to similar enzyme preparations, enabling it to adapt to a wider range of applications. It is not limited to in vitro antibacterial disinfection but also has great potential for in vivo drug applications.

[0024] Compared with traditional antibiotics for treating Helicobacter pylori, this invention has the advantages of being environmentally friendly, having no toxic side effects, no residue risk, and being green and safe. Based on traditional heterologous expression and purification methods, the synthesis of recombinant expression vectors, culture conditions, induction expression methods, and purification methods were optimized. Through multiple experiments, the optimal preparation method was determined to obtain the best yield and purity of the recombinant antibacterial protein.

[0025] Lactiplantibacillus plantarum LP1Z is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 62256. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. The deposit date is February 21, 2022. Attached Figure Description

[0026] Figure 1 This is a prediction chart of the antibacterial function of LP1Z_01335.

[0027] Figure 2 This is an electrophoresis image of recombinant protein LP1Z_01335, where M: protein molecular weight marker, 1: purified protein LP1Z_01335.

[0028] Figure 3 This is a diagram of the conserved domains of the LP1Z_01335 protein.

[0029] Figure 4 The effect of recombinant LP1Z_01335 on the growth of Staphylococcus aureus (G+) and Escherichia coli (G-).

[0030] Figure 5 The effects of recombinant LP1Z_01335 on the cell walls and cell membranes of Staphylococcus aureus (G+) and Escherichia coli (G-).

[0031] Figure 6 The effect of recombinant LP1Z_01335 on the growth of Helicobacter pylori.

[0032] Figure 7 The effect of recombinant LP1Z_01335 on the morphology and structure of Helicobacter pylori.

[0033] Figure 8 It is the stability of recombinant LP1Z_01335 against pH, temperature, and enzymes.

[0034] Figure 9 The effect of recombinant LP1Z_01335 on IL-8 expression in AGS cells. Detailed Implementation

[0035] To make the content, purpose, and advantages of this invention clearer, the technical solution of this invention is described in detail below with reference to specific embodiments. Unless otherwise specified, the reagents involved in the embodiments of this invention are all commercially available products and can be purchased through commercial channels.

[0036] In the following examples, *Lactiplantibacillus plantarum*, standard *Helicobacter pylori* strain, multi-drug resistant *Helicobacter pylori* strain, *Staphylococcus aureus* and *Escherichia coli* were all purchased from Guangdong Microbial Culture Collection Center, address: 5th Floor, Experimental Building, Compound No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China. Among them: the Chinese name of *Lactiplantibacillus plantarum* is *Lactiplantibacillus plantarum* LP1Z, Latin name: *Lactiplantibacillus plantarum* LP1Z, strain deposit number: GDMCC No: 62256; the Chinese name of standard *Helicobacter pylori* strain is *Helicobacter pylori* ATCC 43504, Latin name: *Helicobacter pylori* ATCC 43504; the Chinese name of multi-drug resistant *Helicobacter pylori* strain is *Helicobacter pylori* GZ6B5, Latin name: *Helicobacter pylori* GZ6B5, strain deposit number: GDMCC No: 61571; the Chinese name of *Staphylococcus aureus* is *Staphylococcus aureus* ATCC 6538, Latin name: *Staphylococcus aureus* ATCC 6538; the Chinese name of *Escherichia coli* is *Escherichia coli* ATCC 25922, Latin name: *Escherichia coli* ATCC 25922.

[0037] Example 1: Mining of anti-Helicobacter pylori functional genes through comparative genomics

[0038] 1. Preparation of genomic DNA library

[0039] The genomic DNA library was constructed for Illumina using the AMT Rapid DNA-Seq Kit (CISTRO). Fragmentation and end repair were performed according to the manufacturer's instructions, adapter ligation was carried out with Illumina adapters, size selection and library DNA amplification were performed using magnetic beads. The library was evaluated using an Agilent Bioanalyzer 2100 and a Qubit 3.0 fluorometer. DNA sequencing was performed on the Illumina Nextseq 550 platform using a High Output v2.5 kit. Reads for the microbial genomic DNA library were prepared with the Rapid Barcoding Sequencing Kit (Nanopore) and sequenced on the Nanopore MinION platform.

[0040] Low-quality reads from Illumina sequencing were filtered out using Trimmomatic software (v0.39). Low-quality and short reads from nanopore sequencing were filtered out using Filtlong software (v0.2.0) (https: / / github.com / rrwick / Filtlong). The filtered Illumina and Nanopore reads were then aligned to contiguous groups for de novo assembly using Unicycler software (v0.4.8).

[0041] 2. Genome annotation and comparative genomics analysis

[0042] Pan-genome analysis of the Prokka output was performed using Roary (v3.11.2), with a BLASTP identity cutoff of 95%. The core genome of *Lactobacillus plantarum* LP1Z was generated using Harvest software (v1.1.2), with ATCC 14931 as the reference genome. After core genome alignment, Gubbins were used for recombination analysis and to remove putative recombination regions. Linear sequence comparisons of genomes from different *Lactobacillus plantarum* strains were visualized using Easyfig (v2.2.3).

[0043] Bioinformatics analysis revealed that *Lactobacillus plantarum* LP1Z possesses a specific functional gene, annotated as a hypothetical cell wall hydrolase protein, which we have named LP1Z_01335. This gene contains LysM, PRK13914, and PRK06347 functional domains. The hypothetical protein LP1Z_01335, encoded by this specific functional gene, contains peptidoglycan-binding and cell wall hydrolase-related domains; therefore, it is hypothesized that this hypothetical protein may possess lytic and antibacterial functions. The amino acid sequence of LP1Z_01335, as shown in SEQ ID NO:1, consists of 352 amino acids. The nucleotide sequence of the encoding gene is shown in SEQ ID NO:2.

[0044] 3. Prediction of the antibacterial function of the hypothetical protein LP1Z_01335

[0045] Using CAMP R3 The database (http: / / www.camp3.bicnirrh.res.in / ) was used to search the amino acid sequence of LP1Z_01335 and predict its probability of having antibacterial function. All three algorithms showed that the hypothetical protein LP1Z_01335 has a high probability of possessing antibacterial function. Figure 1 Therefore, the hypothetical protein LP1Z_01335 is considered to be an antibacterial protein, and its antibacterial and bacteriolytic abilities will be verified in the following examples.

[0046] Currently, there are no reports of this antimicrobial protein antagonizing Hp or even multidrug-resistant Hp in China. Therefore, this invention focuses on describing the optimal preparation method of the antimicrobial protein encoded by the LP1Z_01335 gene, its antibacterial effect against multidrug-resistant Hp, and its application scenarios.

[0047] Example 2: Obtaining the antimicrobial protein LP1Z_01335

[0048] 1. Construction of a recombinant expression vector for the antimicrobial protein LP1Z_01335

[0049] When expressing heterologous proteins, mRNA sequence design can improve the efficiency of foreign protein expression. The simplest and most convenient method for mRNA sequence design is codon optimization. Different species and different cells select different synonymous codons (i.e., codon usage frequencies) when encoding the same amino acid, exhibiting their own biases. Generally, in host cells, mRNAs composed of frequently used codons (optimal codons) will have high protein expression. Therefore, by adjusting the synonymous codons of genes according to the host's codon preferences, eliminating rare codons, and optimizing related parameters such as mRNA secondary structure and motif, the goal of improving translation efficiency can be achieved.

[0050] The codons of the antimicrobial protein LP1Z_01335 sequence (SEQ ID NO. 2) in the Lactobacillus plantarum LP1Z genome were optimized. The sequence was input into an online website (https: / / www.vectorbuilder.cn / tool / codon-optimization.html), and the sequence type, expression host, and restriction endonuclease sites to be avoided were selected for optimization. The optimized sequence is shown in SEQ ID NO. 3, and its encoded amino acid sequence is shown in SEQ ID NO. 4. Sequence SEQ ID NO. 3 and plasmid pET28a were sent to BGI Genomics Co., Ltd. in Shenzhen to synthesize a recombinant plasmid. The recombinant strain was constructed by transforming DH5α strain using the heat shock method. The plasmid was plated on LB agar plates (containing 30 μg / ml kanamycin) and incubated overnight at 37°C. Positive colonies were picked, and after PCR detection, sequencing was performed for verification and comparison with the gene nucleotide sequence. The mutation-free recombinant plasmid was then transformed into the BL21 expression strain using the heat shock method.

[0051] 2. Preparation of recombinant antimicrobial protein LP1Z_01335

[0052] (1) IPTG-induced expression of vector fusion protein

[0053] The BL21 expression strain was inoculated into LB liquid medium containing 30 μg / ml kanamycin and a final concentration of 0.5 mM IPTG, and cultured at 37°C with shaking for 4 h to induce fusion protein expression. The culture was centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the cell pellet was resuspended in lysis buffer (1 mol / L NaCl, 50 mmol / L NaH2PO4, 70 mmol / L Na2HPO4 and 50 mmol / L imidazole, pH 7.6–7.8). The cells were then intermittently disrupted using an ultrasonic homogenizer (400 W, 4 sec operation, 8 sec interval, 20 min total). The cell lysate was centrifuged at low temperature and high speed (4°C, 10000 r / min, 20 min), and the pellet was collected.

[0054] (2) renaturation of inclusion body proteins

[0055] Inclusion bodies were washed three times with inclusion body washing buffer (20 mM Tris, 1 mM EDTA, 2 M urea, 1 M NaCl, 1% Triton X-100, pH 8.0) and dissolved in dissolution buffer (20 mM Tris, 5 mM DTT, 8 M urea, pH 8.0). After incubation at 4°C overnight, the solution was centrifuged at 10,000 r / min for 15 min. The resulting solution was serially diluted in buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0) with stirring, and dialyzed overnight in 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0 solution.

[0056] (3) Ni column affinity purification of fusion protein

[0057] The nickel column for protein purification was first equilibrated with lysis buffer. The dialyzed protein solution was then loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column at a flow rate of 0.5 mL / min. The column was washed with lysis buffer at a flow rate of 0.5 mL / min, followed by washing with wash buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min. Finally, the target protein was eluted with elution buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min, and the eluent was collected. The collected protein solution was added to a dialysis bag and dialyzed overnight with PBS.

[0058] After multiple experiments, it was found that the recombinant antimicrobial protein was difficult to obtain using conventional heterologous expression and purification conditions and methods, resulting in low yields and concentrations. Therefore, some conditions and methods were improved based on conventional methods: codon optimization was performed during the construction of the recombinant vector to avoid codon bias affecting translation efficiency. During induced expression, the protein yield was extremely low under conventional conditions, so the conditions were changed to expression in inclusion bodies: the conventional method involves adjusting the final IPTG concentration to 0.1 M and incubating at 18℃ for 48 h, or at 28℃ for 16-24 h; this method was optimized to a final IPTG concentration of 0.5 mM and incubation at 37℃ for 4 h. Refolding of the inclusion bodies is necessary for subsequent purification. Currently, two commonly used refolding methods are dilution refolding and dialysis refolding. Dilution refolding requires setting different refolding conditions, including protein concentration, urea concentration, temperature, redox conditions, the amount of Ttiton X-100 and cyclodextrin added, and refolding time. These conditions are difficult to determine, have a low success rate, and are time-consuming. Therefore, the dialysis refolding method is adopted (as described above).

[0059] The protein was analyzed by 12% SDS-PAGE; its molecular weight was 45 kDa, and its purity was greater than 90%. Figure 2 This improved the activity and yield of the antimicrobial protein LP1Z_01335, thus obtaining the recombinant antimicrobial protein LP1Z_01335.

[0060] Example 3: Sequence analysis of recombinant antimicrobial protein LP1Z_01335

[0061] Analysis revealed that the primary structure of the recombinant antibacterial protein LP1Z_01335 of this invention consists of 352 amino acids, the specific amino acid sequence of which is shown in SEQ ID NO.1. Comparison with sequences in NCBI blastp indicates that the sequence of this protein already exists in the database, but it is not classified as a protein with a specific function; it is only named a LysM domain-containing protein. Comparison of conserved protein domains shows that the structure of LP1Z_01335 consists of the LysM domain (belonging to the LysM superfamily), the PRK13914 domain (belonging to the PRK13914 superfamily), and the PRK06347 domain. Figure 3 ).

[0062] Example 4: Antibacterial activity of recombinant antimicrobial protein LP1Z_01335

[0063] 1. Antagonistic effect of LP1Z_01335 against common foodborne pathogens

[0064] Staphylococcus aureus (G+) and Escherichia coli (G-) in the logarithmic growth phase were inoculated into nutrient broth (10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, pH 7.2) at a volume fraction of 10%. Recombinant antimicrobial protein was added at different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg / mL). The cultures were cultured aerobically at 37°C with shaking for 48 h. The absorbance (OD600) of the cultures was measured at different time points (0, 2, 4, 6, 8, 10, 12, 24, 48 h) to determine the minimum inhibitory concentration (MIC) of the recombinant antimicrobial protein.

[0065] Conductivity shows a linear relationship with the salt content in the solution. The total salt content can be calculated by measuring conductivity, thereby determining the extent of cell contents leaching and the degree of damage to the bacterial cell wall and cell membrane. Staphylococcus aureus and Escherichia coli in the logarithmic growth phase were diluted with PBS in a 10-10 dilution gradient. -6 Recombinant antimicrobial protein was added, and its concentration was adjusted to the minimum inhibitory concentration (MIC). The mixture was incubated aerobically at 37°C with shaking. 0.5 mL samples were taken at different time points (0, 2, 4, 8, and 12 h) and diluted 25-fold. The conductivity of the culture medium was measured, and the total salt content was calculated. A sample without antimicrobial protein was used as a control.

[0066] The results showed that the minimum antibacterial concentration of the recombinant antimicrobial protein against Staphylococcus aureus was 5 μg / mL, and the minimum antibacterial concentration against Escherichia coli was 20 μg / mL. Figure 4 The total salt content of bacterial cultures without recombinant antimicrobial protein remained almost unchanged over 12 hours. After the addition of recombinant antimicrobial protein, the total salt content of the bacterial cultures showed an upward trend, indicating that the recombinant antimicrobial protein LP1Z_01335 caused some damage to the cell walls and cell membranes of both Gram-positive and Gram-negative bacteria. Figure 5 Staphylococcus aureus culture without added recombinant antimicrobial protein showed a slight upward trend after 8 h, which is speculated to be related to cell cycle apoptosis.

[0067] 2. Antagonistic effect of LP1Z_01335 on Helicobacter pylori and multidrug-resistant Helicobacter pylori

[0068] Contains 1×10 7A CFU / mL Helicobacter pylori GZ6B5 bacterial suspension was cultured at a 10% (v / v) inoculum on Helicobacter pylori selective liquid medium (100 mL of Brucella broth, 10 mL of sterile fetal bovine serum, and 1 mL of Helicobacter pylori selective additive, the brand of which was Qingdao Rishui). Different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg / mL) of recombinant antimicrobial protein were added to the medium. The medium was cultured under microaerophilic conditions at 37℃ with shaking for 96 h. The viable bacterial count in the culture medium was measured at different time points (0, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96 h) to determine the minimum inhibitory concentration (MIC) of the recombinant antimicrobial protein.

[0069] Adjust the suspension of Helicobacter pylori (ATCC 43504 and GZ6B5) to achieve a bacterial count of 1×10⁻⁶. 7 CFU / mL, 100 μL of bacterial suspension was added to a Helicobacter pylori selective medium plate (100 mL Helicobacter pylori agar basal medium, 10 mL sterile fetal bovine serum, 1 mL Helicobacter pylori selective additive; both the Helicobacter pylori agar basal medium and the Helicobacter pylori selective additive were from Qingdao Rissui). The plate was then spread onto sterilized Oxford cups, each containing 100 μL of the minimum inhibitory concentration (MIC) of recombinant antimicrobial protein preparation, Lactobacillus plantarum fermentation supernatant, or Lactobacillus plantarum fermentation supernatant adjusted to pH 7. After refrigerating for 6 h, the plates were transferred to a microtrophic environment (5% O2, 10% CO2, 85% N2) at 37°C and incubated for 72 hours. The size of the inhibition zone was observed and measured.

[0070] Helicobacter pylori (ATCC 43504 and GZ6B5) were co-incubated with the minimum inhibitory concentration of recombinant antimicrobial protein at 37°C with shaking for 4 h, with untreated bacteria as a control. The changes in bacterial morphology were observed by scanning electron microscopy and transmission electron microscopy.

[0071] The results showed that the minimum antimicrobial concentration of the recombinant antimicrobial protein against multidrug-resistant Helicobacter pylori GZ6B5 was 2.5 μg / mL. At this concentration, the inhibition zone diameters of the recombinant antimicrobial protein against Hp ATCC 43504 and GZ6B5 were 18.0±0.1 mm and 21.3±0.1 mm, respectively. The antimicrobial effect against multidrug-resistant Hp GZ6B5 was superior to that of the fermentation supernatant of Lactobacillus plantarum LP1Z (17.8±0.1 mm) and the fermentation supernatant adjusted to pH=7 (15.5±0.1 mm). Figure 6 After incubation with recombinant antimicrobial proteins, Helicobacter pylori cells changed from smooth, well-defined, and regularly arranged spiral rods to irregularly arranged, with visible damage to the cell wall and cell membrane. Figure 7 ).

[0072] Example 5: Stability of recombinant antimicrobial protein LP1Z_01335

[0073] Escherichia coli was used as an indicator bacterium (inoculum amount of 10% by volume). Culture medium without recombinant antimicrobial protein was used as a control (NC). Culture medium with minimum inhibitory concentration of recombinant antimicrobial protein (20 μg / mL) was added. The culture was shaken at 37 degrees Celsius under aerobic conditions for 48 h. The absorbance values ​​of different treatment methods and different time points were measured.

[0074] The pH of the recombinant antimicrobial protein was adjusted to 2, 3, 4, 5, 6, 7, 8, and 9 using hydrochloric acid and sodium hydroxide. After incubation at 37°C for 2 h, the pH was adjusted back to 7. 20 μL of the recombinant antimicrobial protein (concentration 200 μg / ml) was added to 180 mL of logarithmic growth phase E. coli culture medium and cultured under aerobic conditions at 37°C with shaking for 48 h. The OD600 of the culture medium was measured at different time points (0, 2, 4, 6, 8, 10, 12, 24, and 48 h) to verify the stability of the enzyme's antimicrobial activity under the influence of pH.

[0075] The recombinant antimicrobial protein was heated to 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ and incubated for 30 minutes. Then, 20 μL of the recombinant antimicrobial protein (concentration 200 μg / ml) was added to 180 mL of E. coli culture medium in the logarithmic growth phase. The medium was cultured under aerobic conditions at 37℃ with shaking for 48 h. The OD600 of the culture medium was measured at different time points (0, 2, 4, 6, 8, 10, 12, 24, and 48 h) to determine the effect of high temperature on the antimicrobial activity of the enzyme.

[0076] Pepsin, trypsin, papain, and proteinase K were added to the recombinant antimicrobial protein at a final concentration of 1 mg / mL, and incubated at 37℃ for 1 h. 20 μL of the recombinant antimicrobial protein (concentration 200 μg / ml) was added to 180 mL of Escherichia coli culture medium in the logarithmic growth phase, and cultured aerobically at 37℃ with shaking for 48 h. The OD600 of the culture medium was measured every two hours to verify its enzyme stability.

[0077] Antibacterial activity retention rate (%) = 100% - [(OD600)] 处理组 - OD600 未处理组 ) / OD600 NC *100%. The treatment group is the group with added recombinant antimicrobial protein that has been adjusted for pH or temperature, or treated with enzymes; the untreated group is the group with added recombinant antimicrobial protein that has not undergone any treatment; NC is the group with no added recombinant antimicrobial protein.

[0078] The recombinant antimicrobial protein exhibits good pH and temperature tolerance and enzyme stability. Compared with the untreated antimicrobial protein formulation, it retains 88.3% or more of its antimicrobial activity within a pH range of 3.0–9.0, with an optimal pH of 7.0; and retains 99.6% of its antimicrobial activity within a temperature range of 37°C–100°C. After treatment with pepsin, trypsin, and papain, the antimicrobial activity is retained at 60.9%, 88.1%, and 99.9%, respectively. Proteinase K has a significant impact on the activity of the recombinant antimicrobial protein, indicating that the antimicrobial protein of this invention has excellent properties, good stability, and high antimicrobial activity. Figure 8 The wide pH and temperature adaptability range allows the recombinant antimicrobial protein to exert antibacterial effects not only in vitro but also in vivo, inhibiting multidrug-resistant Helicobacter pylori and alleviating gastric inflammation.

[0079] Example 6: Application of recombinant antimicrobial protein LP1Z_01335

[0080] The specific applications of recombinant antimicrobial protein LP1Z_01335 are mainly reflected in three aspects: (1) antagonizing the growth and colonization of *Helicobacter pylori* and multidrug-resistant *Helicobacter pylori* in the gastric environment; (2) alleviating gastric inflammation caused by *Helicobacter pylori* and multidrug-resistant *Helicobacter pylori* infection; (3) forming compound enzyme preparations with other components to prevent the intake and infection of exogenous *Helicobacter pylori*. Its specific application methods are as follows:

[0081] (1) Growth and colonization of antagonistic and multidrug-resistant *H. pylori* in the gastric environment:

[0082] Recombinant antimicrobial proteins or their compound enzyme preparations were administered to experimental animals orally or by gavage at a certain dose (e.g., the minimum inhibitory concentration of Hp is 2.5 μg / mL). The eradication of Hp in humans by recombinant antimicrobial proteins was determined by methods such as breath test, antibody assay, and fecal antigen assay. The clearance efficiency of recombinant antimicrobial proteins on Hp in the stomach of experimental animals was determined by methods such as gastric tissue isolation, colony counting, and specific target quantification.

[0083] The effect of recombinant antimicrobial protein on clearing *Helicobacter pylori* in mice was verified using a target-based quantitative method, by applying a certain dose (10... 8 Mice with Helicobacter pylori (Hp) as a control were used. Specific method: Mice were divided into Hp standard strain group (administered only 10 gavage doses). 8 CFU Helicobacter pylori standard strain ATCC 43504, administered 3 times, once every other day via gavage; Hp multidrug-resistant strain group (administered via gavage only 10 times). 8The study included three groups of mice administered Helicobacter pylori GZ6B5 via gavage three times, every other day: a Hp standard strain + antimicrobial protein preparation group (administered Hp standard strain ATCC 43504 via gavage, followed by daily administration of antimicrobial protein preparation for one week); and a multidrug-resistant Hp strain + antimicrobial protein preparation group (administered Hp GZ6B5 via gavage, followed by daily administration of antimicrobial protein preparation for one week). Six mice were included in each group. The concentration of the antimicrobial protein preparation was the minimum in vitro inhibitory concentration (MIC) of the corresponding Hp standard strain ATCC 43504 or Hp GZ6B5, at a dose of 200 μL / kg mouse. Two weeks after the end of gavage, the mice were dissected and stomach tissue was collected. Bacterial DNA was extracted from the mouse stomach tissue and qPCR was performed using Hp-specific primers (5'-TTG AGCATATCCATGAAGTGCG-3', 5'-AGA AAGCGCCACTTGAGCAC-3'). The results indicated that the colonization rate of the standard strain ATCC 43504 (Hp standard strain group) of Helicobacter pylori in mouse gastric tissue was 10. 4 CFU / g, after administration of the recombinant antimicrobial protein preparation (Hp standard strain + antimicrobial protein preparation group), no positive result was detected in the gastric tissue of mice, indicating that the Hp standard strain was completely eliminated; the colonization level of multidrug-resistant Helicobacter pylori GZ6B5 (Hp multidrug-resistant strain group) in the gastric tissue of mice was 10. 3 After oral administration of recombinant antimicrobial protein preparation (Hp multidrug-resistant strain + antimicrobial protein preparation group), the GZ6B5 colonization level in mouse gastric tissue decreased to 10 CFU / g. 2 CFU / g, or recombinant antimicrobial protein preparation, has a strong scavenging effect on multidrug-resistant *Helicobacter pylori* (Hp).

[0084] (2) Relieve gastric inflammation caused by Hp and multidrug-resistant Hp infection:

[0085] The interaction between Helicobacter pylori and gastric epithelial cells leads to an increase in the production of the pro-inflammatory chemokine interleukin-8 (IL-8). Therefore, IL-8 is often used as a marker of host cell inflammatory response. To investigate the alleviation of cellular inflammation caused by Hp infection, especially multidrug-resistant Hp, by recombinant antimicrobial proteins, the expression level of IL-8 in AGS cells was measured. Specific methods: Adherent AGS cells were divided into three groups according to the treatment method: Hp-infected group (AGS cells co-incubated with H. pylori ATCC 43504 or H. pylori GZ6B5 at MOI: 1:100, 37℃ for 6 h), Hp + antimicrobial protein group (AGS cells co-incubated with H. pylori ATCC 43504 or H. pylori GZ6B5 at MOI: 1:100, with the antimicrobial protein concentration adjusted to the minimum inhibitory concentration, cultured at 37℃ for 6 h), and untreated group (AGS cells untreated, cultured normally for 6 h). After culturing, the cell pellet was removed, RNA was extracted and reversed into cDNA, and qPCR fluorescence quantification was performed using IL-8 primers (5'-TTTCTGCAGCTCTCTGTGAGG-3', 5'-CTGCTGTTGTTGTTGCTTCTC-3').

[0086] The results showed that, compared with untreated AGS cells (NC), cells infected with Helicobacter pylori ATCC 43504 or Helicobacter pylori GZ6B5 exhibited a significantly upregulated IL-8 expression level. After the addition of the recombinant antimicrobial protein preparation, the IL-8 expression level was significantly downregulated. Figure 9 This indicates that the presence of the recombinant antimicrobial protein LP1Z_01335 can reduce the expression levels of pro-inflammatory factors such as IL-8 and alleviate gastric inflammation caused by Helicobacter pylori infection.

[0087] (3) Combined with other ingredients to form a compound enzyme preparation product to prevent the intake and infection of exogenous Helicobacter pylori:

[0088] Helicobacter pylori infection is mainly transmitted through oral-oral, fecal-oral, gastro-oral, and iatrogenic routes. Therefore, adding anti-H. pylori active substances to daily oral care products such as toothpaste and mouthwash, as well as cleaning and disinfection products for medical devices, can effectively prevent H. pylori infection. Examples include oral care products containing astragalus, forsythia, scutellaria, licorice, menthol, and antibacterial proteins; breath fresheners containing ovotransferrin, antibacterial proteins, moringa leaf extract, and alfalfa milk extract; and toothpaste containing antibacterial proteins, probiotics, clove leaves, astragalus, and evodia.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

[0090] SEQ ID NO.1 (amino acid sequence of LP1Z_01335)

[0091] MKIKHLLLSSLATTGAIALGATAAKADTVTVKAGDTVSEIADTHKTTVAAVQKLNHLKNVNLIYVGDKLQVNKHQTVKMTRATTSVATPSTTTTTTSAAVQSPTSTVTATSTVSQSTASQASASSVTSAASQSSTSAVSQSSTSQTSASSVTSATSQSTTSAVSQSSTSQASASSV TSAASQSTSTTSQSRTSQASASSVTSTTSQSATSAASQNSTSQASASSVKSAASQSTTVTSQSTTTASSAATSSASTSSTTASQASSSTASSTTTSTTTSSTSTSEAAAKAWIAARESGGSYTVTNGIYYGKYQLGQSLLNGDYSAANQEATADAYVAGRYGSWTAAKAFWQANGWY

[0092] SEQ ID NO.2 (original nucleotide sequence of LP1Z_01335)

[0093]

[0094] SEQ ID NO.3 (nucleotide sequence after codon optimization of LP1Z_01335)

[0095] GATACCGTTACCGTTAAAGCAGGTGATACCGTTAGCGAAATTGCAGATACCCATAAAACCACCGTTGCAGCAGTTCAGAAACTGAATCATCTGAAAAATGTGAATCTGATTTACGTGGGCGACAAGCTGCAGGTTAATAAACATCAGACCGTGAAAATGACCCGCGCAACCACCAGCGTTGCAACCCCTAGTACAACAACCACCACAACCAGTGCAGCAGTTCAAAGTCCGACAAGCACCGTTACCGCAACAAGCACCGTTAGCCAGAGCACCGCAAGCCAGGCAAGTGCAAGTAGTGTTACAAGTGCAGCAAGCCAAAGCAGCACCAGCGCAGTTAGCCAGAGCAGCACAAGTCAGACAAGCGCAAGCAGCGTTACCAGCGCAACCAGCCAAAGTACCACCAGCGCAGTTAGCCAGAGCAGCACCAGTCAGGCAAGCGCAAGTAGCGTTACCAGCGCAGCAAGCCAAAGCACCAGCACCACAAGTCAGAGTCGTACAAGCCAGGCAAGCGCAAGCAGCGTTACAAGCACCACCAGCCAGAGCGCAACCAGCGCAGCAAGTCAAAATAGCACCAGCCAGGCAAGCGCAAGCAGCGTTAAAAGCGCAGCAAGCCAGAGCACCACCGTTACCAGTCAAAGCACCACCACCGCAAGTAGCGCAGCAACCAGTAGTGCAAGCACAAGCAGCACCACCGCAAGCCAGGCAAGTAGCAGTACAGCAAGTAGCACCACCAGCACAACCACCAGCAGTACCAGCACCAGCGAAGCAGCAGCAAAAGCATGGATTGCAGCACGTGAAAGCGGTGGTAGCTATACCGTTACCAATGGTATTTATTACGGCAAGTATCAGCTGGGTCAGAGCCTGCTGAATGGTGATTATAGCGCAGCAAATCAGGAAGCAACCGCAGATGCCTATGTTGCAGGTCGTTATGGTAGCTGGACCGCAGCAAAAGCATTTTGGCAGGCAAATGGTTGGTATTAA

[0096] SEQ ID NO: 4 (Amino acid sequence of LP1Z_01335 after codon optimization)

[0097] DTVTVKAGDTVSEIADTHKTTVAAVQKLNHLKNVNLIYVGDKLQVNKHQTVKMTRATTSVATPSTTTTTTSAAVQSPTSTVTATSTVSQSTASQASASSVTSAASQSSTSAVSQSSTSQTSASSVTSATSQSTTSAVSQSSTSQASASSVTSAASQSTSTTSQSRTSQASASSVTSTTSQSATSAASQNSTSQASASSVKSAASQSTTVTSQSTTTASSAATSSASTSSTTASQASSSTASSTTSTTTSSTSTSEAAAKAWIAARESGGSYTVTNGIYYGKYQLGQSLLNGDYSAANQEATADAYVAGRYGSWTAAKAFWQANGWY

Claims

1. The use of the antimicrobial protein LP1Z_01335, whose amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.4, in any of the following: a. Application in the preparation of drugs for treating Helicobacter pylori infection; b. Application in the preparation of antimicrobial agents, wherein the antimicrobial agents are effective against Staphylococcus aureus, Escherichia coli or Helicobacter pylori.

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