Hydrolase for splitting bacterial peptidoglycan causing chronic inflammatory diseases

Abstract

The invention relates to the technical field of biochemistry, and discloses a hydrolase for splitting bacterial peptidoglycan causing chronic inflammatory diseases, the hydrolase is separated from a human intestinal microbe genome, and the hydrolase can hydrolyze a peptidoglycan layer of gram-positive pathogenic bacteria in a targeted manner, so that the bacterial peptidoglycan has the advantages that the bacterial peptidoglycan has a good anti-inflammatory effect, and the bacterial peptidoglycan has a good anti-inflammatory effect. The compound has high efficiency, specificity and low drug resistance and has a synergistic effect with existing antibiotics; the preparation method of the hydrolase comprises the following steps: according to an amino acid sequence, selecting escherichia coli preferred codons, and artificially synthesizing a gene sequence for coding the hydrolase; cloning the synthetic gene into an expression vector to construct a recombinant expression plasmid; transforming the recombinant expression plasmid into escherichia coli, and carrying out induced expression; the hydrolase is obtained through a separation and purification step. According to the hydrolase for splitting the bacterial peptidoglycan causing the chronic inflammatory disease, a peptidoglycan layer of gram-positive bacteria can be efficiently hydrolyzed through the hydrolase, so that the bacteria are rapidly split, and the antibacterial effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and specifically to a hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases. Background Art

[0002] Antibiotics were once considered the most powerful weapon for treating bacterial infectious diseases. However, with the increasing abuse of antibiotics, the problem of bacterial drug resistance has become increasingly serious, and the speed of new antibiotic research and development is much lower than the speed of the emergence of drug-resistant bacteria.

[0003] Peptidoglycan (PGN) is the main component of the cell wall of Gram-positive bacteria and is crucial for maintaining the cell morphology, size, and survival of bacteria. Hydrolyzing the peptidoglycan of bacteria can help lyse pathogenic bacteria, thereby playing a role in treating or preventing infections caused by the pathogenic bacteria. By mining the genomes of the human gut microbiota, the present invention has discovered a hydrolase with high efficiency, specificity, low drug resistance, and synergistic effects with existing antibiotics, providing a new idea for the research and development of anti-infective drugs. Summary of the Invention

[0004] (I) Technical Problems to be Solved

[0005] Aiming at the deficiencies of the prior art, the present invention provides a hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases, which has the advantages of high efficiency, specificity, low drug resistance, and synergistic effects with existing antibiotics, and solves the problems of strong drug resistance and slow research and development speed of existing antibiotics.

[0006] (II) Technical Solutions

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A hydrolase for cleaving the peptidoglycan of bacteria causing chronic inflammatory diseases, characterized in that: the hydrolase is isolated from the human intestinal microbiota genome and has the following amino acid sequence: SDEDSGTGMSMQTVVQEINTEYDTKLQEEKNSVSYDVLEMSGSRAVWKEVLAVYSVKTNTDQDNPQEVATMDDGKKQLLKDIFWEMNQISSRTESKTETVITETDDGHGNIVETESTVTQTYLYITVSHKTAEEMAAQYGFNEEQKGYLAELLADENNYLWSQVLYGITGGDGQIVTVALSLVGNVGGQPYWSWYGFNSRVEWCACFVSWCANECGYIDAGVIPKYAGCVNGVQWFKDRGQWLDGSAEPAPGMIIFFDWADESGQDGLSDHTGIVQKVENGKVYTVEGNSGDSCRVNEYSIGYYEILGYGAPAY. This hydrolase can target and hydrolyze the peptidoglycan layer of Gram-positive pathogenic bacteria, and has high efficiency, specificity, low drug resistance and a synergistic effect with existing antibiotics.

[0009] As a preferred technical solution of the present invention, the preparation method of the hydrolase includes:

[0010] According to the amino acid sequence, Escherichia coli-preferred codons are selected to artificially synthesize the gene sequence encoding the hydrolase;

[0011] The synthetic gene is cloned into an expression vector to construct a recombinant expression plasmid;

[0012] The recombinant expression plasmid is transformed into Escherichia coli for induced expression;

[0013] The hydrolase is obtained through a separation and purification step.

[0014] As a preferred technical solution of the present invention, the expression vector is the pET22b vector, the recombinant expression plasmid is named pET28a-CHAPPly187, and the Escherichia coli is BL21(DE3) or Origami TM B(DE3) cells.

[0015] As a preferred technical solution of the present invention, the hydrolase can be used to cleave the peptidoglycan layer of Gram-positive pathogenic bacteria causing chronic inflammatory diseases, thereby playing a role in treating or preventing infections caused by the pathogenic bacteria.

[0016] As a preferred technical solution of the present invention, the hydrolase can undergo a hydrolysis reaction with the peptidoglycan of Gram-positive bacteria in vitro to generate detectable hydrolysis products, thereby proving its lytic activity against Gram-positive bacteria.

[0017] As a preferred technical solution of the present invention, the hydrolase can be purified and synthesized on a large scale by biological technology means, reducing production costs and at the same time reducing environmental pollution, and is suitable for industrial production.

[0018] (III) Beneficial effects

[0019] Compared with the prior art, the present invention provides a hydrolase for lysing the peptidoglycan of bacteria causing chronic inflammatory diseases, having the following beneficial effects:

[0020] The hydrolase for lysing the peptidoglycan of bacteria causing chronic inflammatory diseases can efficiently hydrolyze the peptidoglycan layer of Gram-positive bacteria through the hydrolase, thereby rapidly lysing bacteria, improving the antibacterial effect, and the hydrolase is derived from the genome of the human intestinal microbiota, can reduce the interference with normal flora, reduce side effects, and relative to traditional antibiotics, the hydrolase is not easily induced to cause bacterial drug resistance, and the hydrolase has a synergistic effect with existing antibiotics, can enhance the antibacterial effect, broaden the scope of drug application, and the hydrolase can also be purified and synthesized on a large scale by biological technology means, reducing production costs and at the same time reducing environmental pollution. Brief description of the drawings

[0021] Figure 1 It is a diagram of the protein of the present invention containing a CHAP domain;

[0022] Figure 2 It is a diagram showing the hydrolysis of peptidoglycan when the protein of the present invention is co-incubated with peptidoglycan;

[0023] Figure 3 It is a diagram of the computer simulation of the present invention finding that peptidoglycan monomers can bind to the protein. Detailed implementation manners

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0025] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present invention.

[0026] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installed", "connected", and "coupled" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected, or indirectly connected through an intermediate medium, and can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0027] Please refer to Figures 1-3 ,

[0028] A hydrolase for lysing the peptidoglycan of bacteria causing chronic inflammatory diseases, characterized in that: the hydrolase is isolated from the human intestinal microbiota genome and has the following amino acid sequence: SDEDSGTGMSMQTVVQEINTEYDTKLQEEKNSVSYDVLEMSGSRAVWKEVLAVYSVKTNTDQDNPQEVATMDDGKKQLLKDIFWEMNQISSRTESKTETVITETDDGHGNIVETESTVTQTYLYITVSHKTAEEMAAQYGFNEEQKGYLAELLADENNYLWSQVLYGITGGDGQIVTVALSLVGNVGGQPYWSWYGFNSRVEWCACFVSWCANECGYIDAGVIPKYAGCVNGVQWFKDRGQWLDGSAEPAPGMIIFFDWADESGQDGLSDHTGIVQKVENGKVYTVEGNSGDSCRVNEYSIGYYEILGYGAPAY. This hydrolase can target and hydrolyze the peptidoglycan layer of Gram-positive pathogenic bacteria, and has high efficiency, specificity, low drug resistance, and a synergistic effect with existing antibiotics.

[0029] It should be noted that according to the above amino acid sequence, a gene sequence encoding the hydrolase was designed and synthesized. During the synthesis process, Escherichia coli-preferred codons were selected to ensure the high-efficiency expression of the gene in Escherichia coli. The synthesized gene was cloned into the pET22b vector, and the recombinant expression plasmid pET28a-CHAPPly187 was constructed through double digestion (NdeⅠ and HindⅢ) and ligation reactions. The recombinant expression plasmid was sent to a sequencing company for sequencing verification to ensure the correctness of the gene sequence.

[0030] In the present invention, the preparation method of the hydrolase includes:

[0031] According to the amino acid sequence, Escherichia coli-preferred codons were selected, and a gene sequence encoding the hydrolase was artificially synthesized;

[0032] The synthesized gene was cloned into an expression vector to construct a recombinant expression plasmid;

[0033] The recombinant expression plasmid was transformed into Escherichia coli for induced expression;

[0034] Through the separation and purification steps, the hydrolase was obtained.

[0035] It should be noted that the correctly sequenced recombinant expression plasmid was transformed into Escherichia coli BL21(DE3) or Origami TM B(DE3) competent cells to obtain an engineered bacterium expressing the hydrolase. The engineered bacterium was inoculated into an LB medium containing ampicillin and cultured at 37 °C until the OD600 reached 0.6 - 0.8, and then IPTG with a final concentration of 0.5 mM was added for induced expression. The induced expression was carried out at 18 °C for 24 hours to ensure the high-efficiency expression of the hydrolase. After the induced expression was completed, the bacterial cells were collected by centrifugation, and the bacterial cells were disrupted by ultrasonic disruption to release the hydrolase. The hydrolase was separated and purified by methods such as nickel column affinity chromatography to obtain a hydrolase with high purity and high activity.

[0036] In the present invention, the expression vector is the pET22b vector, the recombinant expression plasmid is named pET28a-CHAPPly187, and the Escherichia coli is BL21(DE3) or Origami TM B(DE3) cells.

[0037] In the present invention, the hydrolase can be used to lyse the peptidoglycan layer of Gram-positive pathogenic bacteria causing chronic inflammatory diseases, thereby playing a role in treating or preventing infections caused by the pathogenic bacteria.

[0038] It should be noted that methods such as high performance liquid chromatography (HPLC) are used to detect the hydrolysis activity of hydrolases against peptidoglycan of Gram-positive bacteria; after mixing the hydrolase with peptidoglycan and incubating for a period of time under suitable conditions, the activity of the hydrolase is verified by detecting the generation of hydrolysis products.

[0039] In the present invention, the hydrolase can undergo a hydrolysis reaction with the peptidoglycan of Gram-positive bacteria in vitro to generate detectable hydrolysis products, thereby demonstrating its lytic activity against Gram-positive bacteria.

[0040] In the present invention, the hydrolase can be purified and synthesized on a large scale by biological technology means, reducing production costs and at the same time reducing environmental pollution, and is suitable for industrial production.

[0041] It should be noted that the purified hydrolase is mixed with appropriate excipients to prepare an anti-infective drug preparation; the therapeutic effect of the drug against infections caused by Gram-positive pathogenic bacteria is verified through animal experiments or clinical trials.

[0042] Beneficial effects:

[0043] The hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases can efficiently hydrolyze the peptidoglycan layer of Gram-positive bacteria through this hydrolase, thereby rapidly lysing bacteria, improving the antibacterial effect, and this hydrolase is derived from the genome of the human gut microbiota, can reduce the interference with normal flora, reduce side effects, and relative to traditional antibiotics, this hydrolase is not easily induced to cause bacterial drug resistance, and this hydrolase has a synergistic effect with existing antibiotics, can enhance the antibacterial effect, broaden the scope of drug application, and this hydrolase can also be purified and synthesized on a large scale by biological technology means, reducing production costs and at the same time reducing environmental pollution.

[0044] Working principle:

[0045] The hydrolase of the present invention binds to the peptidoglycan layer in the cell wall of Gram-positive bacteria through its specific amino acid sequence and domain, exerts a specific hydrolysis effect, cuts the peptide chain between the peptidoglycan layers, causes the bacterial cell wall to rupture, and the bacteria lyses and dies. This hydrolase mainly achieves the antibacterial purpose by destroying the bacterial cell wall and is not easily induced to cause bacterial drug resistance.

[0046] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A hydrolase for lysing the peptidoglycan of bacteria causing chronic inflammatory diseases, characterized in that: The hydrolase is isolated from the human intestinal microbiota genome and has the following amino acid sequence: SDEDSGTGMSMQTVVQEINTEYDTKLQEEKNSVSYDVLEMSGSRAVWKEVLAVYSVKTNTDQDNPQEVATMDDGKKQLLKDIFWEMNQISSRTESKTETVITETDDGHGNIVETESTVTQTYLYITVSHKTAEEMAAQYGFNEEQKGYLAELLADENNYLWSQVLYGITGGDGQIVTVALSLVGNVGGQPYWSWYGFNSRVEWCACFVSWCANECGYIDAGVIPKYAGCVNGVQWFKDRGQWLDGSAEPAPGMIIFFDWADESGQDGLSDHTGIVQKVENGKVYTVEGNSGDSCRVNEYSIGYYEILGYGAPAY. This hydrolase can target and hydrolyze the peptidoglycan layer of Gram-positive pathogenic bacteria, and has high efficiency, specificity, low drug resistance, and a synergistic effect with existing antibiotics.

2. The hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases according to claim 1, characterized in that: The preparation method of the hydrolase includes: According to the amino acid sequence, Escherichia coli-preferred codons are selected to artificially synthesize the gene sequence encoding the hydrolase; The synthetic gene is cloned into an expression vector to construct a recombinant expression plasmid; The recombinant expression plasmid is transformed into Escherichia coli for induced expression; The hydrolase is obtained through separation and purification steps.

3. The hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases according to claim 2, wherein: The expression vector is pET22b vector, the recombinant expression plasmid is named pET28a-CHAPPly187, and the Escherichia coli is BL21(DE3) or Origami TM B(DE3) cells.

4. The hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases according to claim 1, characterized in that: The hydrolase can be used to lyse the peptidoglycan layer of Gram-positive pathogenic bacteria that cause chronic inflammatory diseases, thereby playing a role in treating or preventing infections caused by these pathogenic bacteria.

5. A hydrolase for cleaving peptidoglycan of bacteria causing chronic inflammatory diseases according to claim 1, characterized in that: The hydrolase can undergo a hydrolysis reaction with the peptidoglycan of Gram-positive bacteria in vitro to generate detectable hydrolysis products, thereby proving its lytic activity against Gram-positive bacteria.

6. The hydrolase for lysing peptidoglycan of bacteria causing chronic inflammatory diseases according to claim 1, wherein: The hydrolase can be mass-purified and synthesized by biotechnological means, reducing production costs and simultaneously reducing environmental pollution, and is suitable for industrial production.

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