CN112662650B - Bacteriophage lysozyme, gene and application thereof - Google Patents

Abstract

The invention relates to a phage lysozyme, a gene and an application thereof, wherein the amino acid sequence of the lysozyme is shown as SEQ ID NO:1 is shown. The gene for coding the lysozyme is from a metagenome collected from deep sea sediments, the enzyme is subjected to heterologous expression and purification, and the enzyme is detected to have the optimum temperature of 50 ℃, can keep more than 40% of activity within the range of 30-60 ℃, particularly can keep more than 70% of activity within the range of 30-50 ℃ and has a wider action temperature range; the optimum pH value is 7.0, more than 40% of activity can be kept in the pH range of 4.0-9.0, and the pH value has wide action pH range. The lysozyme provided by the invention has broad-spectrum bactericidal activity, can efficiently kill various gram-positive and gram-negative bacteria, provides excellent gene resources and enzyme resources for developing lysozyme type antibacterial products, and has wide application prospects.

Description

Phage lysozyme, gene and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to phage lysozyme, a gene and application thereof.

Background

In recent years, due to abuse of antibiotics, antibiotic-resistant bacteria in the environment and human body have been increasing, and in particular, multi-resistant bacteria having resistance to various antibiotics have become an increasingly serious threat worldwide. Because conventional antibiotics are not effective in killing antibiotic-resistant bacteria, there is an urgent need to find new antibacterial agents to replace antibiotics to kill antibiotic-resistant bacteria. At present, phage lysozyme is one of the hot spots for researchers to develop novel biological bactericides due to its good specificity, biocompatibility and high-efficiency activity in killing bacteria.

The phage lysozyme has the advantages of high bacteria killing efficiency, good specificity, good human biocompatibility, environmental friendliness, difficulty in generating drug-resistant strains, no possibility of causing bacterial resistance and virulence gene propagation and the like, and provides a wide development prospect for researchers to develop new antibacterial drugs, particularly biological agents capable of killing multi-drug-resistant bacteria. In recent years, with the importance of researchers on the research and application of phage lysozyme, more and more lysozyme genes are identified from phage genomes, are prepared and purified by methods such as a prokaryotic expression system and the like, and are applied to the fields of food preservation, aquaculture, medicine and the like.

However, due to the limitation of the laboratory culture technology, more than 99% of phages on the earth are not cultured and identified by the laboratory, and how to search novel lysozyme genes from the uncultured phages becomes a main bottleneck for exploring and utilizing phage lysozyme resources; most of the known lysozyme has the defects of narrow action temperature range, narrow action pH range and the like. Therefore, the skilled person has been working on the search for new bacteriophage lysozymes with superior biological activity.

Disclosure of Invention

The invention aims to provide phage lysozyme, a gene and application thereof, wherein the gene of the lysozyme (named as lysM 2) has the advantages of wide action pH and temperature range, good thermal stability, broad-spectrum bactericidal activity, capability of efficiently killing various gram-positive and gram-negative bacteria, and good application prospect in the aspects of medicine, food preservation, aquaculture and the like.

To this end, in a first aspect, the present invention provides a lysozyme having an amino acid sequence as set forth in SEQ ID NO:1 is shown.

In a second aspect, the invention provides a gene encoding said lysozyme.

Further, the nucleotide sequence of the gene is shown as SEQ ID NO:2, respectively.

In a third aspect, the present invention provides a nucleic acid construct comprising the gene of the invention.

In a fourth aspect, the invention provides a cell expressing the lysozyme according to the invention and/or comprising the gene according to the invention and/or comprising the nucleic acid construct according to the invention.

Further, the cell may be a prokaryotic cell or a eukaryotic cell.

Further, the cells include, but are not limited to: escherichia coli cells, bacillus subtilis cells, lactobacillus cells, actinomycete cells, yeast cells, algal cells, or the like.

In a fifth aspect, the present invention provides a method for producing lysozyme, comprising the steps of: culturing the cell of the invention under conditions that allow expression of the lysozyme; and purifying lysozyme from the resulting culture.

In a sixth aspect, the invention provides the use of said lysozyme in the preparation of a product for the prevention, inhibition or treatment of bacterial infections.

Further, the bacteria include gram-positive bacteria and gram-negative bacteria. In specific embodiments, the bacteria include staphylococcus aureus, bacillus, escherichia coli, pseudomonas putida.

The invention has the beneficial effects that: the invention discloses a phage lysozyme with a novel sequence from a deep-sea unculturable phage genome by using a virus metagenome method. The phage lysozyme has the following advantages: the range of the action temperature is wide, more than 40 percent of activity can be kept within the range of 30-60 ℃, particularly more than 70 percent of activity can be kept within the range of 30-50 ℃, and the thermal stability is good; can keep more than 40% of activity within the range of pH4.0-9.0; has broad-spectrum bactericidal activity and can efficiently kill various gram-positive and gram-negative bacteria. The invention realizes the heterologous expression and purification of the lysozyme, avoids the propagation of other genes of phage which may cause toxicity or resistance in bacteria, has extremely low similarity with the known lysozyme protein sequence, provides excellent gene resources and enzyme resources for developing lysozyme type antibacterial products, and has wide application prospect.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 is an SDS-PAGE pattern of purified lysM2 protein;

FIG. 2 is a graph showing the effect of temperature on the relative enzyme activity of lysM2 protein;

FIG. 3 is a graph showing the effect of pH on the relative enzyme activity of lysM2 protein;

FIG. 4 shows the results of measuring the thermostability of lysM2 protein.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Unless defined otherwise, 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 belongs.

The examples do not indicate specific techniques or conditions, according to techniques or conditions described in literature in the field (for example, refer to molecular cloning, laboratory Manual, third edition, scientific Press, ed by J. SammBruker et al, huang Peitang, et al) or according to the product instructions. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1 acquisition of lysM2 Gene

In this example, deep sea sediment samples were subjected to virus metagenomic sequencing, and a novel phage lysozyme gene lysM2 was analyzed and identified according to the database annotation results. The nucleotide sequence of lysM2 gene is SEQ ID NO:2, the length of the sequence is 1371bp, and the total code is 456 amino acids.

Example 2 clonal expression of lysM2 Gene

1) Using the total DNA of the deep sea sediment sample in example 1 as a template, primers F (SEQ ID NO:3,5 '-GCACATATGATGACACAGAAGCAACTGCCCT-3') and primer R (SEQ ID NO:4,5 '-AGCAAGCTTCTTGCCCTCCAGCTTCTGCT-3') PCR amplifying the said phage lysozyme lysM2 gene, at the same time introducing NdeI and HindIII restriction enzyme sites at its upstream and downstream;

2) Cloning the lysozyme lysM2 gene obtained in the step 1) to a pET22b (+) escherichia coli expression vector to construct a pET22b-lysM2 recombinant expression vector carrying the lysozyme lysM2 gene;

3) Transforming the recombinant pET22b-lysM2 expression vector obtained in the step 2) into an escherichia coli (E.coli) BL21 (DE 3) competent cell by using a chemical transformation method, coating the transformed BL21 (DE 3) strain on an LB solid culture medium containing 10mg/ml of ampicillin and 10% of agar, and culturing for 24 hours at 37 ℃;

4) Selecting BL21 (DE 3) positive clone strains containing recombinant pET22b-lysM2 expression vectors, placing the strains in LB liquid culture medium containing 10mg/ml ampicillin, and performing shake culture at 37 ℃ and 200rpm overnight;

5) The overnight-cultured BL21 (DE 3) -positive clone strain was cultured in the presence of a 1The diluted solution was inoculated into 500ml of LB liquid medium (containing 10mg/ml ampicillin), and cultured at 37 ℃ with shaking at 200rpm until OD ₆₀₀ Reaching 0.6-1.0, isopropyl thio-beta-D-galactoside (IPTG) was added to a final concentration of 5mM, and the induced expression of lysM2 lysozyme was carried out at 20 ℃ over 10 hours.

Example 3 separation and purification of lysM2 lysozyme

The cells obtained by induction expression in example 2 were collected by centrifugation at 15000rpm, washed three times with PBS, and resuspended in lysis buffer (0.3 mol/L NaCl,10mmol/L imidazole, 50mmol/L NaH) ₂ PO ₄ pH 8.0), carrying out crushing and cracking by using an ultrasonic crushing method (power 80%,5s/5s pulse circulation); carrying out high-speed centrifugation at 15000rpm on the cracked suspension, and collecting supernatant; the supernatant and Ni-NTA Agarose were mixed by rotation for 30min, and the recombinant lysM2 lysozyme was purified according to the instructions of the Ni-NTA Agarose kit.

The purified product was analyzed by SDS-PAGE, and the results of the electrophoresis are shown in FIG. 1, and lane 1 is the purified recombinant LysM2 protein, which has a size consistent with the expected molecular weight (51.8 kD).

Example 4 bacteriostatic test

Respectively recovering Staphylococcus aureus (Staphylococcus aureus), bacillus subtilis, escherichia coli (Escherichia coli) and Pseudomonas putida (Pseudomonas putida), selecting single colony, culturing in LB liquid culture medium at 37 deg.C and 200rpm overnight for 8 hr, respectively diluting appropriate amount of bacterial liquid with PBS buffer solution to O _D600 0.1-0.2, and the concentration of the bacterial liquid reaches 10 ⁸ cfu/mL-10 ⁹ cfu/mL。

And (3) uniformly coating 200 mu L of the diluted bacterial liquid on an LB solid culture medium, placing a sterilized Oxford cup, respectively adding 200 mu L of the lysozyme lysM2 protein (the concentration is 1 mg/mL) prepared in the example 3 into the Oxford cup, culturing in a constant-temperature incubator at 37 ℃ for about 12h, observing and measuring the size of a bacteriostatic zone. The results are shown in table 1:

TABLE 1 bacteriostatic effect of Lysozyme lysM2 on test strains

As can be seen from the bacteriostatic results, lysozyme lysM2 has broad-spectrum bactericidal activity and better bacteriostatic effect on various gram-positive and gram-negative bacteria, wherein the bacteriostatic effect on gram-positive bacteria is better than that of gram-negative bacteria.

Staphylococcus aureus is selected as a substrate, and relative enzyme activities of lysozyme lysM2 at different pH values (pH =3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0) and different temperatures (30 ℃, 40 ℃,50 ℃, 60 ℃ and 70 ℃) are determined according to the method, and the results are shown in fig. 2 and fig. 3, and fig. 2 shows that the optimum action temperature of lysozyme lysM2 is 50 ℃, the activity of more than 40% can be kept in the range of 30 ℃ to 60 ℃, particularly the activity of more than 70% can be kept in the range of 30 ℃ to 50 ℃, and the wide action temperature range is achieved. The optimum action pH value of lysozyme lysM2 is 7.0, and can keep more than 40% of activity within the pH range of 4.0-9.0, and has wide action pH range.

And (3) testing thermal stability: after the lysozyme lysM2 solution is respectively placed at 30 ℃, 40 ℃,50 ℃, 60 ℃ and 70 ℃ for heat preservation for 2 hours, the relative enzyme activity is detected according to the method, and the relative enzyme activity of the enzyme without heat preservation is defined as 100%. As shown in FIG. 4, the detection results show that LysM2 has good thermal stability, and maintains more than 60% of relative enzyme activity after being kept at 50 ℃ for 2h as shown in FIG. 4.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

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Ser Asn Tyr Pro Arg Ala Val Pro Pro Ser Thr Ile Ser Pro Ala Tyr

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Ser Gly Gly Thr Val Tyr His Tyr Met Thr Val Met Gly Tyr Asp Asp

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Glu Gln Arg Ser Val Trp Ile Ala Asp Ser Gly Phe Ser Pro Phe Gly

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Tyr Ala Phe Ala Asp Val Glu Pro Ala Ala Ala Pro Ala Pro Gly Pro

180 185 190

Ala Pro Arg Gly Met Asp Ala Asp Ala Leu Ser Gln Ala Met Gly Gly

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Thr Val Ser Arg Glu Arg Tyr Ala Ala Leu Leu Pro Ala Phe Thr Glu

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Claims (11)

1. The lysozyme is characterized in that the amino acid sequence of the lysozyme is shown in SEQ ID NO:1 is shown.

2. A gene encoding the lysozyme of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is as set forth in SEQ ID NO:2, respectively.

4. A nucleic acid construct comprising the gene of claim 2 or 3.

5. A cell expressing the lysozyme of claim 1, and/or comprising the gene of claim 2 or 3, and/or comprising the nucleic acid construct of claim 4.

6. The cell of claim 5, wherein the cell is a prokaryotic cell or a eukaryotic cell.

7. The cell of claim 6, wherein the cell is an E.coli cell, a B.subtilis cell, a Lactobacillus cell, an actinomycete cell, a yeast cell, or an algal cell.

8. A method for producing lysozyme is characterized by comprising the following steps: culturing the cell of any one of claims 5-7 under conditions that allow expression of the lysozyme of claim 1; and purifying lysozyme from the resulting culture.

9. Use of the lysozyme of claim 1 in the manufacture of a product for the prevention, inhibition, or treatment of a bacterial infection.

10. The use of claim 9, wherein the bacterium is a gram positive bacterium or a gram negative bacterium.

11. The use of claim 10, wherein the gram-positive bacterium is staphylococcus aureus or bacillus, and the gram-negative bacterium is escherichia coli or pseudomonas putida.

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