CN113563427B - Antibacterial short peptide LBD-S and application and medicine thereof - Google Patents
Antibacterial short peptide LBD-S and application and medicine thereof Download PDFInfo
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Abstract
The invention provides an antibacterial short peptide LBD-S and application and a medicament thereof, relating to the technical field of biology. The invention provides an antibacterial short peptide LBD-S, the amino acid sequence of which is CTKKVKPDLKRFEKYFKGTVTC, the research of the inventor finds that the antibacterial short peptide LBD-S has low hydrophobicity, strong hydrophile lipophile and high antibacterial activity, has stronger inhibiting effect on gram negative bacteria and gram positive bacteria, such as bacillus subtilis, corynebacterium glutamicum, micrococcus lytica, micrococcus luteus, photobacterium mermairei and the like, and compared with the antibacterial short peptide LBD-ALF before modification, the antibacterial short peptide LBD-S provided by the invention has obviously enhanced inhibiting effect on the bacillus subtilis, the corynebacterium glutamicum and the micrococcus lytica, and can be used for preparing antibacterial products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibacterial short peptide LBD-S and application and a medicament thereof.
Background
The problem of biological antibiotic resistance is one of the major problems affecting public health and clinical practice, Antimicrobial peptides (AMPs) have attracted considerable attention as an important antibiotic substitute, and most research work is concerned with the identification of Antimicrobial peptides, in vitro activity and mode of action, and microbial resistance mechanisms. The Lipopolysaccharide Binding Domains (LBD) of the anti-lipopolysaccharide factors (ALFs) are the major functional elements of ALF, have antibacterial activity, typically have 22 amino acid residues and consist of two cysteines constituting a disulfide bond structure. Moreover, LBD is considered as a functional domain of antimicrobial and antiviral activity.
Because the activity of a plurality of natural antibacterial peptides identified and separated at present is not ideal or certain toxicity exists on eukaryotic cells, the design and modification of the antibacterial peptides become the first choice for overcoming the defects, and the development of attractive novel antibacterial peptides in the future is facilitated. The natural antibacterial peptide is taken as a base, and the sequence of the natural antibacterial peptide is properly increased or decreased and the base substitution or modification of amino acid is carried out, so that the biological activity of the basic antibacterial peptide is improved, and the natural antibacterial peptide is more suitable for being applied to production practice.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
It is a first object of the present invention to provide an antibacterial short peptide LBD-S to solve at least one of the above problems.
The second purpose of the invention is to provide the application of the antibacterial short peptide LBD-S in the preparation of antibacterial products.
The third purpose of the invention is to provide a medicine which comprises the antibacterial short peptide LBD-S.
In a first aspect, the invention provides an antibacterial short peptide LBD-S, wherein the amino acid sequence of the antibacterial short peptide LBD-S is CTKKVKPDLKRFEKYFKGTVTC.
As a further technical scheme, a disulfide bond structure is formed between the amino-terminal cysteine and the carboxyl-terminal cysteine of the antibacterial short peptide LBD-S.
As a further technical scheme, the amino terminal and the carboxyl terminal of the antibacterial short peptide LBD-S are respectively and independently modified.
As a further technical scheme, the amino terminal of the antibacterial short peptide LBD-S is subjected to acetylation modification;
and the carboxyl terminal of the antibacterial short peptide LBD-S is subjected to amidation modification.
In a second aspect, the invention provides an application of an antibacterial short peptide LBD-S in preparing an antibacterial product.
As a further aspect, the product comprises a medicament or a formulation.
As a further aspect, the bacteria comprise gram positive bacteria and/or gram negative bacteria.
As a further technical scheme, the gram-positive bacteria comprise at least one of bacillus subtilis, corynebacterium glutamicum, micrococcus muralis or micrococcus luteus.
As a further technical solution, the gram-negative bacteria include mermaid photobacterium.
In a third aspect, the invention provides a medicament comprising an antibacterial short peptide LBD-S.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an antibacterial short peptide LBD-S, the amino acid sequence of which is CTKKVKPDLKRFEKYFKGTVTC, the research of the inventor finds that the antibacterial short peptide LBD-S has low hydrophobicity, strong hydrophile lipophile and high antibacterial activity, and compared with the antibacterial short peptide LBD-ALF before modification, the antibacterial short peptide LBD-S provided by the invention has obviously enhanced inhibition effect on bacillus subtilis, corynebacterium glutamicum and micrococcus lyticus and can be used for preparing antibacterial products for gram-negative bacteria (such as bacillus subtilis, corynebacterium glutamicum, micrococcus lyticus and the like) and gram-positive bacteria (such as Photobacterium mermairei and the like).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the arrangement analysis of the hydrophilic and hydrophobic amino acids of LBD-S short peptide;
FIG. 2 is a reversed phase high performance liquid chromatogram of LBD-S.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The design of natural antibacterial peptide as basic template and substituting amino acid in one or several sequences and synthesizing analog is one of the methods for modifying antibacterial peptide. The factors affecting the antibacterial activity of the antibacterial peptide mainly include: net charge, hydrophobicity, amphiphilicity (hydrophobic moment), etc. By adjusting the influencing factors, the activity of the antibacterial peptide derivative is improved.
In a first aspect, the invention provides an antibacterial short peptide LBD-S, wherein the amino acid sequence of the antibacterial short peptide LBD-S is CTKKVKPDLKRFEKYFKGTVTC.
The invention provides an antibacterial short peptide LBD-S, the amino acid sequence of which is CTKKVKPDLKRFEKYFKGTVTC, the research of the inventor finds that the antibacterial short peptide LBD-S has low hydrophobicity, strong hydrophile lipophile and high antibacterial activity, has stronger inhibiting effect on gram negative bacteria and gram positive bacteria, such as bacillus subtilis, corynebacterium glutamicum, micrococcus lytica, micrococcus luteus, photobacterium mermairei and the like, and compared with the antibacterial short peptide LBD-ALF before modification, the antibacterial short peptide LBD-S provided by the invention has obviously enhanced inhibiting effect on the bacillus subtilis, the corynebacterium glutamicum and the micrococcus lytica, and can be used for preparing antibacterial products.
As a further technical scheme, a disulfide bond structure is formed between the amino-terminal cysteine and the carboxyl-terminal cysteine of the antibacterial short peptide LBD-S.
The amino terminal and the carboxyl terminal of the antibacterial short peptide LBD-S provided by the invention are cysteine, and the cysteine at the two terminals can be connected by a disulfide bond, so that the stability of the antibacterial short peptide LBD-S is improved, and the degradation is avoided.
As a further technical scheme, the amino terminal and the carboxyl terminal of the antibacterial short peptide LBD-S are respectively and independently modified.
The stability of the antibacterial short peptide LBD-S can be improved and the degradation of the antibacterial short peptide LBD-S can be slowed down by modifying the amino terminal and the carboxyl terminal of the antibacterial short peptide LBD-S.
As a further technical scheme, the amino terminal of the antibacterial short peptide LBD-S is acetylated and modified.
In the invention, the stability of the amino terminal of the antibacterial short peptide LBD-S can be improved, the degradation rate is slowed down, and the antibacterial action time of the antibacterial short peptide LBD-S is prolonged by performing acetylation modification on the amino terminal of the antibacterial short peptide LBD-S.
And the carboxyl terminal of the antibacterial short peptide LBD-S is subjected to amidation modification.
In the invention, the stability of the carboxyl terminal of the antibacterial short peptide LBD-S can be improved, the degradation rate is slowed down, and the antibacterial action time of the antibacterial short peptide LBD-S is prolonged by performing acetamization modification on the carboxyl terminal of the antibacterial short peptide LBD-S.
In a second aspect, the invention provides an application of an antibacterial short peptide LBD-S in preparing an antibacterial product.
The research of the inventor finds that the antibacterial short peptide LBD-S provided by the invention has low hydrophobicity, strong hydrophily and lipophilicity and high antibacterial activity, and has stronger inhibiting effect on both gram-negative bacteria and gram-positive bacteria, so that the antibacterial short peptide LBD-S can be used for preparing products for resisting gram-negative bacteria and gram-positive bacteria.
As a further technical solution, the product includes, but is not limited to, a drug or a preparation, or other products described by those skilled in the art, such as a feed additive or a feed including the feed additive, and the like.
As a further aspect, the bacteria comprise gram positive bacteria and/or gram negative bacteria.
The inventor finds that the antibacterial short peptide LBD-S has stronger inhibiting effect on both gram-negative bacteria and gram-positive bacteria.
As a further aspect, the gram-positive bacteria include, but are not limited to, at least one of Bacillus subtilis, Corynebacterium glutamicum, Micrococcus muralis, or Micrococcus luteus.
As a further aspect, the gram-negative bacteria include, but are not limited to, photobacterium mermairei.
In a third aspect, the present invention provides a medicament, including but not limited to the antibacterial short peptide LBD-S, which may further include pharmaceutically acceptable excipients, etc.
The antibacterial short peptide LBD-S provided by the invention has high antibacterial activity and stronger inhibiting effect on gram-negative bacteria and gram-positive bacteria, so that the medicine comprising the antibacterial short peptide LBD-S also has all effects of the antibacterial short peptide LBD-S.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
EXAMPLE 1 engineering of antimicrobial peptides LBD short peptides based on MjALF-D
According to The Analysis of The physicochemical properties and The structural information of The LBD short Peptide of MjALF-D, The Analysis Software uses The ProtParam online Analysis Software (http:// www.expasy.org/tools/ProtParam) in The Exterprotein Analysis Software (ExPASY) website and The antibacterial Peptide Database (APD, http:// APs. unmac. edu/AP/main. php) online Analysis Software to obtain The relevant parameters of The polypeptide. Analysis of the arrangement of the hydrophilic and hydrophobic amino acids of the short peptides and helical wheel projection were performed using the online analysis software Heliquest analysis (http:// helix. ipmc. cnrs. fr/cgi-bin/ComputParams. py) (Gautier et al, 2008). And (2) carrying out sequence comparison on the LBD short peptide (LBD-ALF) of MjALF-D in an APD (avalanche photo diode) antibacterial peptide system, and carrying out amino acid substitution according to the comparison result and all obtained related parameters, thereby improving the activity of the short peptide and modifying and designing to obtain the novel antibacterial short peptide LBD-S.
The amino acid sequences of LBD-ALF and LBD-S are as follows:
LBD-ALF：CTYNVKPDLQRFELYFLGTVTC(SEQ ID NO.1)；
LBD-S：CTKKVKPDLKRFEKYFKGTVTC(SEQ ID NO.2)。
the new antibacterial short peptide LBD-S is based on LBD-ALF, and lysine (K) is used to replace tyrosine (Y), asparagine (N), glutamine (Q) and leucine (L). The physicochemical properties and amino acid arrangement of the antibacterial short peptide LBD-S are shown in Table 1 and figure 1, and compared with LBD-ALF, the net charge amount is increased (+5), the hydrophobicity is obviously reduced, and the amphipathicity is obviously enhanced.
TABLE 1 analysis of physicochemical Properties of LBD short peptides
EXAMPLE 2 Artificial Synthesis of antibacterial short peptide LBD-S short peptide
The antibacterial LBD short peptide which is designed by engineering is synthesized. The synthesis method is based on solid phase chemical synthesis, the synthesis direction is from the C end to the N end of the LBD-S short peptide, acetylation modification is carried out on the N end, and amidation modification is carried out on the C end, so as to play a role in enhancing the stability of the synthesized polypeptide. The carboxyl and amino termini of LBD-S are cysteines, forming disulfide bonds. The synthesized LBD-S short peptide was purified by reversed-phase high performance liquid chromatography and the purity thereof was examined, and the results are shown in FIG. 2.
As can be seen from FIG. 2, the synthesized antibacterial short peptide LBD-S has only one prominent peak and has high purity (95.0749%).
EXAMPLE 3 MIC and MBC assay of antibacterial short peptide LBD-S
The detection and analysis of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the protein are carried out by adopting a liquid growth inhibition determination method, and the specific determination method refers to the following documents:
[1]Ying P,Libing Z,Mao Y,et al.The antibacterial activity and mechanism analysis of piscidin 5 like from Larimichthys crocea[J].2019,92:43-49.
the results are shown in Table 2.
TABLE 2 MIC and MBC (unit: μ M) for LBD-ALF and modified LBD-S short peptides
Note: NT: not detected.
As can be seen from Table 2, the modified antibacterial short peptide LBD-S has good inhibitory action on gram-positive bacteria (including Bacillus subtilis, Corynebacterium glutamicum, Micrococcus muralyticus and Micrococcus luteus) and gram-negative bacteria (including Photobacterium mermairei), and has significantly enhanced inhibitory action on Bacillus subtilis, Corynebacterium glutamicum, Micrococcus muralyticus and Micrococcus luteus compared with LBD-ALF before modification.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Zhuhai school district of Beijing university of teachers and professions
<120> antibacterial short peptide LBD-S and application and medicament thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> PRT
<213> Artificial sequence
<400> 1
Cys Thr Tyr Asn Val Lys Pro Asp Leu Gln Arg Phe Glu Leu Tyr Phe
1 5 10 15
Leu Gly Thr Val Thr Cys
20
<210> 2
<211> 22
<212> PRT
<213> Artificial sequence
<400> 2
Cys Thr Lys Lys Val Lys Pro Asp Leu Lys Arg Phe Glu Lys Tyr Phe
1 5 10 15
Lys Gly Thr Val Thr Cys
20
Claims (10)
1. An antibacterial short peptide LBD-S, wherein the amino acid sequence of the antibacterial short peptide LBD-S is CTKKVKPDLKRFEKYFKGTVTC.
2. The antibacterial short peptide LBD-S according to claim 1, wherein said antibacterial short peptide LBD-S has a disulfide bond structure between the amino-terminal cysteine and the carboxyl-terminal cysteine.
3. The antibacterial short peptide LBD-S according to claim 1, wherein the amino terminus and the carboxy terminus of said antibacterial short peptide LBD-S are independently modified.
4. The antibacterial short peptide LBD-S according to claim 2, wherein the amino terminal of the antibacterial short peptide LBD-S is modified by acetylation;
and the carboxyl terminal of the antibacterial short peptide LBD-S is subjected to amidation modification.
5. Use of the antibacterial short peptide LBD-S according to any one of claims 1 to 4 for the preparation of an antibacterial product.
6. Use according to claim 5, wherein the product comprises a medicament or a formulation.
7. Use according to claim 5, wherein the bacteria comprise gram-positive and/or gram-negative bacteria.
8. The use of claim 7, wherein the gram-positive bacteria comprise at least one of Bacillus subtilis, Corynebacterium glutamicum, Micrococcus muralyticus, or Micrococcus luteus.
9. Use according to claim 7, wherein the gram-negative bacteria comprise Photobacterium mermairei.
10. A medicament comprising the antibacterial short peptide LBD-S according to any one of claims 1 to 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1218057A (en) * | 1997-09-29 | 1999-06-02 | 遗传和生物技术工程中心 | New immunological activity for peptide of limulus anti-LPS factor |
| US6384188B1 (en) * | 1993-08-18 | 2002-05-07 | Dana-Farber Cancer Institute, Inc. | Lipopolysaccharide-binding and neutralizing peptides |
| CN104248751A (en) * | 2013-06-28 | 2014-12-31 | 东吴大学 | Anti-cancer peptide, use and pharmaceutical composition containing anti-cancer peptide |
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| US20020107174A1 (en) * | 1998-07-02 | 2002-08-08 | Erik J. Paus | Composition comprising endotoxin neutralizing protein and derivatives and uses thereof |
| JP2002541457A (en) * | 1999-04-07 | 2002-12-03 | ユニリーバー・ナームローゼ・ベンノートシヤープ | Lipopolysaccharide immunoassay and test equipment |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6384188B1 (en) * | 1993-08-18 | 2002-05-07 | Dana-Farber Cancer Institute, Inc. | Lipopolysaccharide-binding and neutralizing peptides |
| CN1218057A (en) * | 1997-09-29 | 1999-06-02 | 遗传和生物技术工程中心 | New immunological activity for peptide of limulus anti-LPS factor |
| CN104248751A (en) * | 2013-06-28 | 2014-12-31 | 东吴大学 | Anti-cancer peptide, use and pharmaceutical composition containing anti-cancer peptide |
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