CN112679594A

CN112679594A - Modified analgesic peptide BRL of antibacterial peptide Buforin IIB and application thereof

Info

Publication number: CN112679594A
Application number: CN202011595594.7A
Authority: CN
Inventors: 孙同毅; 高媛媛; 张赛; 罗禄军
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-04-20
Anticipated expiration: 2040-12-29
Also published as: CN112679594B

Abstract

The invention provides a modified analgesic peptide BRL of antibacterial peptide Buforin IIB and application thereof. The amino acid sequence of the analgesic peptide BRL is shown as SEQ ID NO.1 and is obtained by modifying the amino acid sequence of antibacterial peptide Buforin IIB; the analgesic peptide BRL has remarkable antibacterial activity, analgesic activity and anti-inflammatory activity, and has simple structure and convenient acquisition. The analgesic peptide BRL can be applied to preparation of a medicine for treating or relieving pain or a medicine for treating or relieving inflammation, and therefore has a good clinical application prospect.

Description

Modified analgesic peptide BRL of antibacterial peptide Buforin IIB and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a modified analgesic peptide BRL of antibacterial peptide Buforin IIB and application thereof.

Background

The antibacterial peptide is a natural small molecular polypeptide encoded by organism genes and is also an important molecule of an organism immune system. The antibacterial peptide Buforin II has broad-spectrum and efficient bactericidal capacity on bacteria (G +/G-) and fungi, and has stronger bactericidal effect on the fungi compared with the bacteria, which is also a unique advantage compared with other Buforin analogues. Buforin II can also inhibit membrane fusion by inhibiting the formation of SNARE complex, effectively inhibit neurotransmitter release, and can be used for treating skin wrinkle and nervous diseases caused by excessive neurotransmitter release. Researchers can obtain the antibacterial peptide Buforin IIB with stronger antibacterial effect by splicing fragments of different polypeptides with a Buforin core sequence for modification.

At present, the polypeptide is taken as a molecule with biological activity, so that the polypeptide is widely concerned and applied in the medical fields of disease treatment, tissue repair and the like, and the artificially synthesized polypeptide is used for modifying biological materials, so that the immune risk of biological source protein is avoided, the toxic and side effects are low, the biological activity is high, the compatibility is good, and the development of the polypeptide as a novel polypeptide analgesic drug becomes a new trend.

Disclosure of Invention

The invention aims to provide a modified analgesic peptide BRL of antibacterial peptide Buforin IIB and application thereof. The analgesic peptide BRL has obvious analgesic activity and anti-inflammatory activity, and is simple in structure and convenient to prepare.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a modified analgesic peptide BRL of antibacterial peptide Buforin IIB, and the amino acid sequence of the analgesic peptide BRL is shown as SEQ ID No. 1.

Furthermore, the analgesic peptide BRL has significant analgesic activity, anti-inflammatory activity and bacteriostatic activity.

Further: the analgesic peptide BRL is obtained by modifying an amino acid sequence of antibacterial peptide Buforin IIB.

Further: the amino acid sequence of the antibacterial peptide Buforin IIB is RAGL QFPLG RLLR RLLR RLLR.

The invention also provides application of the modified analgesic peptide BRL of the antibacterial peptide Buforin IIB in preparation of a medicine for treating or relieving pain.

Further, the pain includes pain caused by inflammation, pain caused by high temperature or thermal stimulation, abdominal pain, and acute pain.

Furthermore, the concentration of the analgesic peptide BRL is 5-10 mg/ml.

Further, the analgesic peptide BRL can remarkably relieve pain.

Furthermore, the analgesic peptide BRL can remarkably relieve acute pain and ankylosing spondylitis.

The invention also provides application of the modified analgesic peptide BRL of the antibacterial peptide Buforin IIB in preparation of anti-inflammatory drugs.

Furthermore, the analgesic peptide BRL can obviously inhibit inflammation.

The invention also provides a medicament for resisting inflammation or/and easing pain, which contains the modified analgesic peptide BRL of the antibacterial peptide Buforin IIB.

Compared with the prior art, the invention has the advantages and the technical effects that:

the invention relates to a novel analgesic peptide BRL obtained by transforming antibacterial peptide Buforin IIB, the amino acid sequence of the analgesic peptide BRL is searched and compared by a protein database, no identical polypeptide is found, and the analgesic peptide BRL has the advantages of simple structure and good analgesic effect. The invention verifies that the analgesic peptide BRL has obvious analgesic effect and anti-inflammatory activity through experiments, shows good analgesic and anti-inflammatory effects in a mouse pain experiment animal model experiment and a carrageenan foot swelling experiment, has wide application range, can be used for preparing anti-inflammatory or/and analgesic drugs, and has simple structure, convenient acquisition and wide clinical application prospect compared with other analgesic peptides.

Drawings

FIG. 1 shows the results of the time to first writhing of mice after BRL injection.

FIG. 2 shows the results of acetic acid writhing frequency of mice within 20 minutes after the injection of analgesic peptide BRL.

FIG. 3 shows the results of one phase of formalin test mice after injection of analgesic peptide BRL.

FIG. 4 shows two-phase results of formalin test mice after BRL injection.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1

1. Obtention of analgesic peptide BRL

According to the amino acid sequence (RAGLQFPLGRLLRRLLRRLLR) of the antibacterial peptide Buforin IIB, the antibacterial peptide Buforin IIB is modified, then the antibacterial peptide Buforin IIB is synthesized by a solid-phase synthesis method, and then analgesic activity screening is respectively carried out by using an acetic acid writhing experiment, a formalin model experiment and a carrageenan model experiment, so that the antibacterial peptide Buforin IIB modified analgesic peptide BRL with analgesic activity is obtained.

2. Molecular identification of analgesic peptide BRL

(1) Determination of amino acid sequence:

the polypeptide crude product synthesized by the artificial solid phase is purified by high performance liquid chromatography to obtain analgesic peptide BRL, and the complete amino acid sequence of the analgesic peptide BRL is determined by Edman degradation method on a full-automatic protein sequence determinator, and the result shows that the amino acid sequence of the analgesic peptide BRL is RLLRRLLRRLLR (SEQ ID NO. 1).

(2) Mass spectrometry molecular weight determination:

and (3) detecting the accurate molecular weight of the purified analgesic peptide BRL by matrix-assisted laser desorption time of flight mass spectrometry, wherein the used instrument is an AutoflexiiiTOF/TOF mass spectrometer of Bruker company. The results show that the molecular weight of analgesic peptide BRL is 1634.10Da by mass spectrometry.

Example 2: analgesic Activity assay for analgesic peptide BRL

In order to deeply understand the analgesic activity of the analgesic peptide BRL, the invention utilizes a classic acetic acid writhing animal pain model, a formalin animal pain model and a carrageenan animal pain model to carry out research, and the result shows that the analgesic peptide BRL shows better analgesic activity in the three animal pain models, which are respectively as follows:

1. analgesic effect of analgesic peptide BRL on acetic acid writhing pain model of mouse

The experimental animals were SPF-grade Kunming mice (weight 18-22g) of 4 weeks old. Mice were randomly divided into control group (sterile saline), positive drug group (50mg/kg aspirin), and BRL group (50mg/kg analgesic peptide BRL), each of which was 10 mice. Analgesic peptide BRL was dissolved in sterile physiological saline to a dilution concentration of 5 mg/ml. Injecting 5mg/ml analgesic peptide BRL diluent 200 μ l into left side of mice in BRL group; injecting equal volume of 50mg/kg aspirin into the left abdominal cavity of the positive drug group mouse; the control mice were injected intraperitoneally with an equal volume of sterile saline left side. After 30 minutes, the right side of three groups of mice was intraperitoneally injected with 250. mu.l of 0.7% acetic acid solution, and the time for the mice to develop writhing for the first time after acetic acid injection and the number of writhing within 20 minutes were recorded, respectively.

As shown in fig. 1 and fig. 2, the analgesic peptide BRL can prolong the time of the first writhing of the mouse and reduce the acetic acid writhing frequency of the mouse, and shows that the analgesic peptide BRL has an obvious analgesic effect on the pain of the animal caused by acetic acid.

2. Experiment of analgesic peptide BRL on formalin-induced pain

Mice were randomly divided into control group (sterile saline), positive drug group (50mg/kg aspirin), and BRL group (50mg/kg analgesic peptide BRL), each of which was 10 mice. Analgesic peptide BRL was dissolved in sterile physiological saline to a dilution concentration of 5 mg/ml. Injecting 200 μ l of analgesic peptide BRL with concentration of 5mg/ml into abdominal cavity of mice in BRL group; injecting isovolumic 50mg/kg aspirin into abdominal cavity of positive drug group mouse; control mice were injected intraperitoneally with an equal volume of sterile saline. After 30min of intraperitoneal injection, 20 μ L of 3% formalin solution was injected into the right hind paw of the mouse, and the time for licking the feet was recorded for one phase (0-5min) and two phases (15-30 min).

As shown in fig. 3-4, the experimental results show that the analgesic peptide BRL can significantly reduce the foot licking time of one phase and two phases of the mice, and the time is lower than that of the aspirin group, which indicates that the analgesic peptide BRL not only has a significant analgesic effect on acute pain, but also has a better analgesic effect on ankylosing pain.

3. Carrageenan foot swelling test

The anti-inflammatory effect of the analgesic peptide BRL was evaluated by carrageenan induced swelling of the mouse feet. Mice were randomly divided into a normal group, a model group (carrageenan), a positive group (10mg/kg indomethacin), and a BRL group (50mg/kg analgesic peptide BRL), each of which was 10 mice. Analgesic peptide BRL was dissolved in sterile physiological saline to a dilution concentration of 5 mg/ml. Injecting sterile normal saline into the abdominal cavity of the mice of the normal group and the model group, injecting 5mg/ml analgesic peptide BRL 200 mu l into the mice of the BRL group, and injecting 10mg/kg indometacin into the mice of the positive group; 30min after the intraperitoneal injection of the drug, the right hind paw of the normal group of mice was injected with 30 μ l of physiological saline, the model group was injected with 30 μ l of 1% carrageenan, the positive group and the BRL group of mice were injected with 30 μ l of 1% carrageenan, respectively, and the right hind paw thickness of the mice was measured every other hour from 2 h.

The experimental results (table 1) show that the carrageenan mouse model is successfully established by comparing the control group data with the model group data, the model group data and the positive group data reach the highest peak of the swelling degree at 3h, compared with the two groups, the analgesic peptide BRL has the inflammation inhibition effects at different degrees at 2h, 3h, 4h, 5h and 6h, wherein the inhibition effect at 3h is most obvious, which indicates that the analgesic peptide BRL has an obvious anti-inflammatory effect.

Table 1: experimental results for swelling of foot in carrageenan-model mice

In conclusion, the invention obtains the new modified analgesic peptide BRL by modifying the amino acid sequence of the antibacterial peptide Buforin IIB, and verifies that the analgesic peptide BRL not only has obvious analgesic effect, but also has obvious anti-inflammatory effect through various mouse pain experimental animal model experiments, has wide application range, can be used for treating or relieving inflammation and pain caused by inflammation, pain caused by high temperature or thermal stimulation, abdominal pain, acute pain and other various pains, and can be used for preparing the medicines for treating or relieving pain.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Weifang medical college

<120> modified analgesic peptide BRL of antibacterial peptide Buforin IIB and application thereof

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<170> SIPOSequenceListing 1.0

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Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg

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<213> antibacterial peptide (Buforin IIB)

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Arg Ala Gly Leu Gln Phe Pro Leu Gly Arg Leu Leu Arg Arg Leu Leu

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Claims

1. A modified analgesic peptide BRL of antibacterial peptide Buforin IIB is characterized in that: the amino acid sequence of the analgesic peptide BRL is shown in SEQ ID NO. 1.

2. The modified analgesic peptide BRL of the antimicrobial peptide Buforin IIB of claim 1, wherein: the analgesic peptide BRL has significant analgesic activity, anti-inflammatory activity and bacteriostatic activity.

3. The modified analgesic peptide BRL of the antimicrobial peptide Buforin IIB of claim 1, wherein: the analgesic peptide BRL is obtained by modifying an amino acid sequence of antibacterial peptide Buforin IIB.

4. Use of the modified analgesic peptide BRL of the antimicrobial peptide Buforin IIB as claimed in any one of claims 1 to 3 in the manufacture of a medicament for the treatment or alleviation of pain.

5. The use of the modified analgesic peptide BRL of the antimicrobial peptide Buforin IIB as claimed in claim 4 in the manufacture of a medicament for the treatment or alleviation of pain, wherein: the pain includes pain caused by inflammation, pain caused by high temperature or thermal stimulation, abdominal pain, and acute pain.

6. The use of the modified analgesic peptide BRL of the antimicrobial peptide Buforin IIB as claimed in claim 4 in the manufacture of a medicament for the treatment or alleviation of pain, wherein: the concentration of the analgesic peptide BRL is 5-10 mg/ml.

7. Use of the modified analgesic peptide BRL of the antibacterial peptide Buforin IIB as claimed in any one of claims 1 to 3 in the manufacture of a medicament for anti-inflammatory use.

8. The use of the modified analgesic peptide BRL of the antimicrobial peptide Buforin IIB as claimed in claim 7 in the manufacture of a medicament for anti-inflammatory therapy, wherein: the analgesic peptide BRL can obviously inhibit inflammation.

9. A medicament for anti-inflammation or/and analgesia, which is characterized by comprising modified analgesic peptide BRL of antibacterial peptide Buforin IIB.