Broad-spectrum antibacterial lyase as well as preparation and application thereof

Abstract

The invention provides engineering lyase Art-15 with broad-spectrum antibacterial activity, antibacterial peptide NZ2114 and bacteriophage lyase PlySs2 are subjected to functional fusion through an optimally designed flexible linker, and an efficient bactericidal effect on Staphylococcus aureus (Staphylococcus aureus) and Streptococcus spp.) is achieved. The engineering lyase Art-15 has the advantages that the engineering lyase Art-15 has broad-spectrum antibacterial activity, the antibacterial peptide NZ2114 and the bacteriophage lyase PlySs2 are subjected to functional fusion through the optimally designed flexible linker, and the engineering lyase Art-15 has broad-spectrum antibacterial activity; the lyase Art-15 overcomes the defects of narrow host spectrum and insufficient splitting activity of the lyase through a synergistic destruction mechanism of targeting bacterial cell wall peptidoglycan. Experiments show that the minimal inhibitory concentration (MIC) of the lyase Art-15 on staphylococcus aureus in vitro is lower than 2 mu g / mL, the minimal inhibitory concentration of the lyase Art-15 on streptococcus agalactiae is lower than 1 mu g / mL, the minimal inhibitory concentration of the lyase Art-15 on streptococcus dysgalactiae is lower than 2 mu g / mL, and the sterilization rate of the lyase Art-15 can reach 97% or above within 30 minutes by 2 times of MIC. The lyase Art-15 disclosed by the invention has important application value in the fields of drug-resistant bacterium infection treatment, medical instrument disinfection and livestock breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a lyase with broad-spectrum antibacterial activity, its preparation and application. Background Art

[0002] Bacterial resistance has become a major challenge in the field of global public health, severely limiting effective anti-infective treatment methods. Most clinically used antibiotics act by inhibiting enzymes in the following metabolic pathways, such as peptidoglycan synthesis, ribosomal protein synthesis, folic acid synthesis, and nucleic acid synthesis and topoisomerization. Although these antibiotics can inhibit or kill bacteria, on the other hand, they also prompt bacteria to develop resistance. In recent years, biomolecular antibacterial strategies have attracted increasing attention, including phage therapy and phage-derived proteins. Phage therapy has host specificity. However, compared with small molecule antibiotics, the large size of phages poses pharmacokinetic challenges and may pose a risk of inducing phage-neutralizing antibodies.

[0003] Compared with the large size of phages, phage-derived proteins (such as lyases) have attracted increasing attention in recent years. They kill bacteria by degrading the peptidoglycan layer in the cell wall. Recent studies have explored methods to optimize the activity of phage lyases through protein engineering means, such as fusing antimicrobial peptides (AMPs) or cell-penetrating peptides (CPPs) to phage lyases to enhance their killing ability against pathogenic bacteria. When designing fusion proteins, linker peptides are usually used to integrate two functional domains or proteins. The rigidity and length of the linker peptide can affect the activity of the fusion protein, and when a certain degree of movement or interaction is required between the two linked functional domains, a flexible linker is usually used. However, most of the obtained lyases only inhibit a single bacterial species. For example, patent CN202010146580.0 discloses a fusion antibacterial protein that has a bactericidal effect on Staphylococcus aureus. However, Streptococcus bacteria such as Streptococcus agalactiae, which are common in the field of animal health and certain human infectious diseases and are opportunistic pathogens, have very limited antibacterial reports. Therefore, the development of a new type of antibacterial agent that can simultaneously cover multiple pathogenic bacteria, has high antibacterial efficiency and is resistant to the environment has become a key breakthrough point in solving drug-resistant bacterial infections in the medical and animal health fields. Summary of the Invention

[0004] In view of this, the present invention provides a novel engineered lyase Art-15, which is linked to the antimicrobial peptide NZ2114 and a phage lyase through a linker, has broad-spectrum antibacterial activity, can efficiently kill Staphylococcus aureus and Streptococcus bacteria, and has broad application prospects in antibacterial drugs.

[0005] The technical solution of the present invention is realized as follows:

[0006] In a first aspect, a broad-spectrum antibacterial lyase Art-15 is provided, which comprises an antibacterial peptide NZ2114 functional domain, a flexible linker, and a lyase PlySs2 sequentially connected from the N-terminus to the C-terminus. The amino acid sequence of the lyase Art-15 is as shown in SEQ ID NO: 2 or has at least 95% homology.

[0007] On the basis of the above solution, the lyase PlySs2 is derived from a Streptococcus suis (S. suis) phage; the sequence of the flexible linker is (GGGGS)3.

[0008] On the basis of the above solution, a gene encoding the above-mentioned lyase Art-15 is provided, and its nucleotide sequence is as shown in SEQ ID NO: 1 or a variant with improved expression efficiency after codon optimization.

[0009] On the basis of the above solution, a recombinant expression vector is provided, which comprises the above-mentioned lyase Art-15 gene.

[0010] On the basis of the above solution, a recombinant strain is provided, which comprises the above-mentioned recombinant expression vector.

[0011] In a second aspect, a method for preparing the above-mentioned broad-spectrum antibacterial lyase Art-15 is provided, comprising the following steps:

[0012] S1, constructing a lyase Art-15 fusion gene. Preferably, gene fragments encoding the antibacterial peptide NZ2114, the linker, and PlySs2 are amplified and sequentially ligated by overlap extension PCR amplification to construct a fusion gene;

[0013] S2, cloning the fusion gene into an expression vector to obtain a recombinant plasmid. Preferably, the expression vector is pET-28a(+), which contains elements such as a promoter, a ribosome binding site, a multiple cloning site, and a resistance gene;

[0014] S3, transforming the recombinant plasmid into a host cell to obtain a recombinant bacterium. Preferably, the host cell is Escherichia coli BL21(DE3);

[0015] S4, culturing the recombinant bacterium to induce the expression of lyase Art-15. Preferably, the induction conditions are 0.1 mM IPTG and induction at 15 °C for 12 h;

[0016] S5, purifying the lyase Art-15. Preferably, Ni-NTA or Ni-TED is used for purification.

[0017] In a third aspect, the above-mentioned broad-spectrum antibacterial lyase Art-15 is provided for use in the preparation of an antibacterial pharmaceutical composition.

[0018] On the basis of the above solution, the dosage form of the antibacterial drug composition is one of injection, nebulizer, oral liquid and lyophilized powder;

[0019] On the basis of the above solution, the application of the antibacterial drug composition in the preparation of drugs for treating Gram-positive bacterial infections. Preferably, the Gram-positive bacteria include Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae.

[0020] On the basis of the above solution, the application of the antibacterial drug composition as an antibacterial agent for the surface of instruments, an animal feed additive, a veterinary drug, or a food antibacterial agent.

[0021] The broad-spectrum antibacterial lyase Art-15 of the present invention has the following beneficial effects compared with the prior art:

[0022] (1) In the present invention, the antibacterial peptide NZ2114 and the phage lyase PlySs2 are functionally fused through an optimized flexible linker. Based on the synergistic destruction mechanism targeting the peptidoglycan of the bacterial cell wall, the defects of narrow host spectrum and insufficient lytic activity of the lyase are overcome;

[0023] (2) The broad-spectrum antibacterial lyase Art-15 of the present invention significantly improves its antibacterial performance by fusing antibacterial peptides, has excellent antibacterial activity, can exert effective antibacterial effects at extremely low concentrations, has a minimum inhibitory concentration (MIC) against Staphylococcus aureus in vitro lower than 2 μg / mL, a minimum inhibitory concentration against Streptococcus agalactiae lower than 1 μg / mL, and a minimum inhibitory concentration against Streptococcus dysgalactiae lower than 2 μg / mL. Moreover, a bactericidal rate of more than 97% can be achieved within 30 minutes at 2-fold MIC, realizing efficient bactericidal effects against Staphylococcus aureus and Streptococcus spp.;

[0024] (3) The antibacterial drug composition containing the broad-spectrum antibacterial lyase Art-15 of the present invention can provide new solutions for the treatment of drug-resistant bacterial infections, the disinfection of medical devices, and the prevention and treatment of animal infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 This is the structural schematic diagram of Art-15 of the present invention;

[0027] Figure 2 This is the nucleic acid electrophoresis diagram of Art-15 of the present invention;

[0028] Figure 3 This is the physicochemical property analysis diagram of Art-15 of the present invention;

[0029] Figure 4 This is the antibacterial activity determination diagram of Art-15, NZ2114 and PlySs2 of the present invention against Staphylococcus aureus GRNSTA2402003;

[0030] Figure 5 This is the antibacterial activity determination diagram of Art-15, NZ2114 and PlySs2 of the present invention against Streptococcus agalactiae GRNSAG2408001;

[0031] Figure 6 This is the antibacterial activity determination diagram of Art-15, NZ2114 and PlySs2 of the present invention against Streptococcus dysgalactiae GRNSD2402001. Detailed implementation manners

[0032] Next, in combination with the implementation manners of the present invention, the technical solutions in the implementation manners of the present invention will be clearly and completely described. Obviously, the described implementation manners are only a part of the implementation manners of the present invention, rather than all of the implementation manners. Based on the implementation manners in the present invention, all other implementation manners obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0033] Example 1:

[0034] Construction of engineered lyase Art-15

[0035] The antibacterial peptide NZ2114, flexible linker (GGGGS)3 and lyase PlySs2 fragments were fused by the overlap extension PCR method. The specific process is as follows:

[0036] Primers were designed to ensure that the primers carried overlapping sequences for subsequent fragment connection. In the first overlap extension PCR, the gene fragments of NZ2114 and (GGGGS)3 were used as templates, and a forward primer (targeting the 5' end of NZ2114 and carrying the overlapping sequence of the pET28a(+) vector) and a reverse primer (targeting the 3' end of (GGGGS)3 and carrying the overlapping sequence of the 3' end of NZ2114) were used. The fusion fragment of NZ2114-(GGGGS)3 was obtained by PCR amplification and connection. The amplification conditions are shown in Table 1.

[0037] Table 1 Overlap extension PCR amplification conditions

[0038]

[0039]

[0040] The product was purified after being verified by agarose gel electrophoresis. Then, in the second overlap extension PCR, the first PCR products NZ2114-(GGGGS)3 and PlySs2 fragments were used as templates, and the same forward primer and a new reverse primer (targeting the 3' end of PlySs2 and carrying the overlapping sequence of the pET28a(+) vector) were used. The same PCR reaction conditions were repeated to ligate NZ2114-(GGGGS)3 with PlySs2, and finally, a 903-bp NZ2114-(GGGGS)3-PlySs2 fusion gene was formed, as Figure 2 (lanes 3, 4, and 5) shown in SEQ ID NO.1.

[0041] GGTTTCGGCTGTAACGGTCCGTGGAACGAGGACGATCTGCGTTGCCACAACCACTGCAAGAGCATCAAAGGTTACAAAGGTGGCTATTGCGCTAAAGGTGGTTTCGTTTGCAAATGCTATGGTGGTGGCGGCTCTGGTGGTGGTGGTTCCGGCGGTGGTGGCTCCATGACTACCGTTAACGAAGCTCTGAACAACGTTCGTGCTCAGGTTGGTTCTGGCGTTTCTGTGGGTAACGGCGAGTGCTACGCACTGGCCAGCTGGTACGAACGTATGATCTCTCCGGATGCTACCGTTGGTCTGGGTGCTGGTGTAGGCTGGGTTTCTGGCGCGATTGGTGACACTATCTCCGCTAAGAACATCGGCAGCTCTTACAACTGGCAGGCTAACGGTTGGACCGTTTCCACTTCCGGTCCATTCAAAGCTGGCCAGATCGTGACTCTGGGTGCTACTCCAGGCAACCCGTATGGTCACGTTGTTATCGTCGAGGCAGTAGACGGTGATCGTCTGACCATCTTGGAACAGAACTACGGTGGTAAACGCTATCCAGTTCGTAACTACTACTCTGCGGCGAGCTACCGTCAGCAGGTTGTTCACTACATCACCCCGCCGGGTACTGTTGCTCAGTCCGCACCAAACCTGGCTGGTTCTCGTTCTTACCGTGAGACTGGTACTATGACCGTTACCGTTGATGCACTGAACGTGCGTCGTGCGCCGAACACCTCCGGTGAGATCGTAGCCGTTTACAAACGTGGTGAGTCTTTCGACTACGATACCGTTATCATCGATGTCAACGGTTACGTTTGGGTGTCTTACATCGGCGGTTCTGGTAAACGTAACTACGTTGCTACTGGCGCAACTAAAGACGGCAAACGTTTCGGCAACGCGTGGGGCACCTTCAAGTAA

[0042] The protein sequence encoded by this fusion gene is: NZ2114-Linker-PlySs2, as shown in SEQ ID NO.2.

[0043] GFGCNGPWNEDDLRCHNHCKSIKGYKGGYCAKGGFVCKCYGGGGSGGGGSGGGGSMTTVNEALNNVRAQVGSGVSVGNGECYALASWYERMISPDATVGLGAGVGWVSGAIGDTISAKNIGSSYNWQANGWTVSTSGPFKAGQIVTLGATPGNPYGHVVIVEAVDGDRLTILEQNYGGKRYPVRNYYSAASYRQQVVHYITPPGTVAQSAPNLAGSRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVIIDVNGYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK

[0044] Subsequently, the fusion gene was cloned into the BamHI / SalI site of the expression vector pET28a(+) using Gibson assembly, and the assembly reaction conditions are shown in Table 2. The recombinant plasmid was named pET28a-Art-15. After being verified correct by sequencing, the constructed recombinant plasmid was transformed into the Escherichia coli BL21(DE3) host bacterium for subsequent expression and purification experiments.

[0045] Table 2 Gibson assembly reaction conditions

[0046]

[0047] Example 2:

[0048] Expression and purification of engineered lyase Art-15

[0049] Positive clones were picked on an LB plate medium containing 50 mg / L kanamycin and inoculated into an LB medium (50 mg / L kanamycin) and cultured until the OD 600 reached 0.6 - 0.8. IPTG with a final concentration of 0.1 mM was added for induction of expression, and induction was continued for 12 hours under low-temperature culture at 15 °C to improve the solubility of the protein. After the culture was completed, the bacterial cells were collected and lysed by sonication, and then His-tag purification was performed using a Ni 2+ affinity chromatography column. The eluted protein was dialyzed to remove impurities, and SDS-PAGE was used to analyze the purity and molecular weight of the protein. At the same time, a Nanodrop instrument was used to verify the expression level of the fusion protein, and finally, highly pure Art-15 lyase was obtained.

[0050] Example 3:

[0051] Physicochemical properties of engineered lyase Art-15

[0052] To explore the physicochemical properties of the engineered lyase Art-15, we analyzed the amino acid sequence of the Art-15 protein using the ExPASy ProtParam tool. By submitting the sequence of Art-15 online, multiple key parameters were obtained, and the results are as Figure 3 shown.

[0053] Analysis showed that the molecular weight of Art-15 was 31.4 kDa and the isoelectric point (pI) was 8.96. Amino acid composition analysis indicated that its Aliphatic Index was 64.63, suggesting that Art-15 had thermal stability. In addition, its Grand Average of Hydropathicity (GRAVY) was -0.301. Compared with the wild-type lyase PlySs2 (-0.230), Art-15 was more hydrophilic overall, less likely to form inclusion bodies, and beneficial for production and purification. The instability index was 26.16, lower than 40, indicating that the protein was stable in solution.

[0054] The above results indicated that the physicochemical properties of Art-15 conferred potential advantages in higher temperature environments, aqueous solution systems, and production processes, providing a theoretical basis for subsequent functional verification and optimization.

[0055] Example 4:

[0056] Determination of the minimum inhibitory concentration of the engineered lyase Art-15

[0057] To further quantify the antibacterial ability of Art-15, the present invention used the microbroth dilution method to determine its minimum inhibitory concentration (MIC). In the experiment, Art-15 was serially diluted 2-fold in TSB or TSB + 5% horse blood medium in a 96-well plate, and inoculated with approximately 10 5 CFU / mL of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae suspensions, and incubated at 37 °C for 16 - 18 hours. After incubation, the OD of each well was measured by colorimetry 600Values were determined, and the lowest concentration at which Art-15 completely inhibited bacterial growth was identified. The experimental results showed that the MIC values of Art-15 against Staphylococcus aureus were in the range of 0.5 - 2 μg / mL, those against Streptococcus dysgalactiae were in the range of 0.125 - 2 μg / mL, and those against Streptococcus agalactiae were even lower, in the range of 0.125 - 1 μg / mL (Table 3), demonstrating excellent antibacterial activity. This indicates that Art-15 can exert effective antibacterial effects at extremely low concentrations, laying a foundation for its application in the fields of medical treatment and prevention and control of animal infections. In addition, compared with the antimicrobial peptide NZ2114 and the wild-type lyase PlySs2, Art-15 showed more excellent antibacterial ability. The engineering strategy of the lyase (enhancing its synergistic bactericidal effect by fusing the antimicrobial peptide) significantly improved its antibacterial performance, providing an effective design idea for the development of new antibacterial agents.

[0058] Table 3 Minimum inhibitory concentrations of Art-15, NZ2114, and PlySs2 against Staphylococcus aureus and Streptococcus spp.

[0059]

[0060]

[0061] Example 5:

[0062] Determination of antibacterial activity of engineered lyase Art-15

[0063] The bactericidal activities of Art-15, NZ2114, and PlySs2 against Staphylococcus aureus strain GRNSTA2402003, Streptococcus agalactiae GRNSAG2408001, and Streptococcus dysgalactiae GRNSD2402001 were determined by the plate counting method. Art-15, NZ2114, and PlySs2 were serially diluted 2-fold in TSB or TSB + 5% bovine serum medium to different concentrations, and 10 6 CFU / mL bacteria were added and incubated at 37 °C for 30 minutes. 100 μL of the culture solution was taken and spread on TSB or TSB + 5% horse blood agar plates, and the number of colonies was counted after incubation at 37 °C for 20 hours. The antibacterial activity of Art-15 was evaluated by calculating the bactericidal rate. The results showed that Art-15 had a more significant bactericidal effect against Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae compared with the antimicrobial peptide NZ2114 and the wild-type lyase PlySs2, as shown in Figure 4 , Figure 5 and Figure 6 respectively.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A lytic enzyme Art-15 with broad-spectrum antibacterial activity, characterized in that, The lyase Art-15 described above comprises an antibacterial peptide NZ2114 functional domain, a flexible linker, and a lyase PlySs2, which are sequentially connected from the N-terminus to the C-terminus; the amino acid sequence of the lyase Art-15 is as shown in SEQ ID NO:

2.

2. The lyase Art-15 according to claim 1, characterized in that, The lyase PlySs2 is derived from a Streptococcus suis (S.suis) phage, and the sequence of the flexible linker is (GGGGS)3.

3. A gene encoding the lyase Art-15 as claimed in claim 1, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO:

1.

4. A recombinant expression vector, characterized in that, It contains the lyase Art-15 gene as described in claim 3.

5. A recombinant strain, characterized in that, It contains the recombinant expression vector as described in claim 4.

6. A preparation method of the broad-spectrum antibacterial lyase Art-15 as described in claim 1, characterized in that, It includes the following steps: S1, constructing a lyase Art-15 fusion gene; S2, cloning the fusion gene into an expression vector to obtain a recombinant plasmid; S3, transforming the recombinant plasmid into a host cell to obtain a recombinant bacterium; S4, culturing the recombinant bacterium to induce the expression of the lyase Art-15; S5, purifying the lyase Art-15.

7. The preparation method according to claim 6, characterized in that, The expression vector is pET-28a(+), and the host cell is Escherichia coli BL21(DE3).

8. An antibacterial pharmaceutical composition, characterized in that, It contains the lyase Art-15 as described in claim 1, and the dosage form of the antibacterial drug composition is one of injection, nebulizer, oral liquid, and lyophilized powder.

9. The application of the antibacterial drug composition as described in claim 8 in the preparation of a drug for treating Gram-positive bacterial infections, wherein the Gram-positive bacteria include Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae.

10. The application of the antibacterial drug composition as described in claim 8 as a surface antibacterial agent for instruments, an animal feed additive, a veterinary drug, or a food antibacterial agent.

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