CN112851752B - Human milk endogenous antibacterial polypeptide and application thereof in preparation of anti-inflammatory drugs - Google Patents
Human milk endogenous antibacterial polypeptide and application thereof in preparation of anti-inflammatory drugs Download PDFInfo
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- CN112851752B CN112851752B CN201911190593.1A CN201911190593A CN112851752B CN 112851752 B CN112851752 B CN 112851752B CN 201911190593 A CN201911190593 A CN 201911190593A CN 112851752 B CN112851752 B CN 112851752B
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 50
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 37
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 31
- 235000020256 human milk Nutrition 0.000 title claims abstract description 18
- 210000004251 human milk Anatomy 0.000 title claims abstract description 18
- 230000000844 anti-bacterial effect Effects 0.000 title claims description 17
- 238000002360 preparation method Methods 0.000 title description 6
- 229940124599 anti-inflammatory drug Drugs 0.000 title description 4
- 241000588724 Escherichia coli Species 0.000 claims abstract description 13
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 11
- 235000013305 food Nutrition 0.000 claims abstract description 5
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
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- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- KTFZQPLSPLWLKN-KKUMJFAQSA-N Pro-Gln-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KTFZQPLSPLWLKN-KKUMJFAQSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- SLCSPPCQWUHPPO-JYJNAYRXSA-N Tyr-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 SLCSPPCQWUHPPO-JYJNAYRXSA-N 0.000 description 1
- DWAMXBFJNZIHMC-KBPBESRZSA-N Tyr-Leu-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O DWAMXBFJNZIHMC-KBPBESRZSA-N 0.000 description 1
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- 238000006460 hydrolysis reaction Methods 0.000 description 1
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- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
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- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a polypeptide compound YEKYLGPQYV which is derived from human milk endogenous source and has inhibitory activity on gram-positive bacteria and gram-negative bacteria represented by escherichia coli and staphylococcus aureus, and the amino acid sequence of the polypeptide compound is Tyr-Glu-Lys-Tyr-Leu-Gly-Pro-Gln-Tyr-Val. The polypeptide YEKYLGPQYV has broad inhibitory activity on gram-positive bacteria and gram-negative bacteria, and has good application prospect as a medicament and/or health product for preventing and/or reducing intestinal infection diseases of newborns caused by microorganisms or an additive of novel infant formula food.
Description
Technical Field
The invention relates to an endogenous antibacterial polypeptide YEKYLGPQYV derived from human milk and application thereof.
Background
Antibiotics are drugs that can resist pathogenic microorganisms, and are the largest class of antibacterial and anti-inflammatory drugs. Antibiotics are substances produced by bacteria, fungi or other microorganisms in the life process, have the effect of inhibiting or killing pathogenic microorganisms such as bacteria and fungi, and are widely applied to various infectious diseases as anti-infective drugs. The significant effects of antibiotics in the treatment of infections also lead to bacterial resistance, making the treatment of some infectious diseases increasingly difficult. The antibacterial peptide is used as a novel antibiotic, has broad-spectrum antibacterial activity, can not easily cause the drug resistance of microorganisms, and has wide application prospect.
Antibacterial peptides are widely distributed in nature, and as a novel antibiotic which is widely concerned, more and more antibacterial peptides are found in microorganisms, animals and plants. The structure-activity relationship of the currently analyzed antibacterial peptide has two clear characteristics, namely 'positively charged cation' and 'amphiphilic sequence structure'. However, antibacterial peptides still face a number of problems to be solved as antibacterial drugs: part of the cationic antibacterial peptide can produce larger toxic effect on cells of mammals; the amphiphilic structure requires a certain amount of basic amino acids in an amino acid sequence, and the characteristic that the basic amino acids are easily hydrolyzed by protease ensures that the effect of the basic amino acids is greatly limited after the basic amino acids are used as medicines and enter a human body. Therefore, the development of safe, stable and low-cytotoxicity antibacterial peptides is of great significance.
The breast milk is the best natural food for infants, is rich in protein and polypeptide resources, and has the advantages of the antibacterial peptide and the high-quality and safe protein source of the human milk. Human milk contains a variety of immune-related proteins. Endogenous enzyme in human mammary gland has stronger hydrolysis effect on protein, and is derived from endogenous antibacterial peptide stably existing in human milk, the problem that the antibacterial peptide is easy to be further hydrolyzed after entering human body is solved due to the endogenous enzyme, and the antibacterial peptide has good potential as an antibacterial medicament. Therefore, a new treatment means for clinically treating infant infection resistance is provided by further searching for peptides with bacteriostatic activity from human milk endogenous polypeptides.
Disclosure of Invention
The invention aims to provide an application of endogenous antibacterial polypeptide YEKYLGPQYV in preparation of medicines and/or health-care products or novel infant formula foods for preventing and/or reducing intestinal infection diseases caused by escherichia coli and staphylococcus aureus.
In order to achieve the above purpose, the present invention uses the polypeptide YEKYLGPQYV as an effective component for inhibiting the growth activity of microorganisms.
It has the sequence table of SEQ ID NO: 1, amino acid sequence; the polypeptide YEKYLGPQYV is an active ingredient of medicine for resisting Escherichia coli and Staphylococcus aureus, and can be added with pharmaceutically acceptable carrier or adjuvant.
The amino acid sequence of the polypeptide compound YEKYLGPQYV with inhibitory activity to escherichia coli and staphylococcus aureus is Tyr-Glu-Lys-Tyr-Leu-Gly-Pro-Gln-Tyr-Val. The molecular weight is 1259.42Da, the white powder is easily dissolved in water, and the compound has strong inhibition effect on the growth of escherichia coli and staphylococcus aureus.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains and determines the structure of the polypeptide from human milk, and verifies that the polypeptide has better bacteriostatic activity for the first time, so the polypeptide has good application prospect as a medicament and/or health-care product for preventing and/or reducing intestinal infection diseases of newborns caused by microorganisms or an additive of novel infant formula food.
Drawings
FIG. 1 shows the bacteriostatic results of polypeptide YEKYLGPQYV, wherein A is the experimental result of polypeptide YEKYLGPQYV on inhibiting Escherichia coli, and B is the experimental result of polypeptide YEKYLGPQYV on inhibiting Staphylococcus metalens.
Detailed Description
Example 1
Preparation and identification of human milk endogenous peptides.
(1) Sample preparation
Diluting 200 μ L of human milk stock solution with water 10 times, denaturing in water bath at 95 deg.C for 5min, ultrafiltering with ultrafiltration tube with molecular weight cut-off of 10K Da at 14000 Xg and 20 deg.C for 20min, and collecting ultrafiltrate as endogenous peptide.
(2)Triple TOFTM6600+ MS analysis
Endogenous peptide samples were desalted on a C18 column (Waters Oasis HLB SPE column) as follows: commercial C18 column was activated with 1.5mL of methanol and equilibrated with 1.5mL of 0.1% (V/V) TFA-H2O solution, the sample was redissolved with 0.1% (V/V) TFA-H2O solution and loaded onto C18 column, eluted with 1.5mL of 80% (V/V) ACN/0.1% (V/V) TFA-H2O solution, the eluate was collected and lyophilized for storage at-80 ℃. Samples of endogenous peptide obtained from the above procedure were reconstituted with 0.1% (V/V) FA-H2O at a sample concentration of 0.4mg/ml, all samples were loaded in the same volume of 8. mu.L, and Triple TOF was performedTM6600+ MS analysis, 3.2. mu.g of sample. NanoLC-MS/MS analysis was performed in an Information Dependent Acquisition (IDA) mode using a Triple TOF 6600 mass spectrometer (AB SCIEX, USA). Pretreatment column C18, analytical column: CHROMXP C18,3UM,150X0.3MM COLUMN. Tandem mass spectra are acquired in positive ion data-dependent mode with the mass scan set to a full scan charge-to-mass ratio (m ≧ greater than or equal to { }z) ions in the range of 350-1250, the accumulation time is 0.25s, the strongest 40 ions are subjected to ion collision dissociation (CID), and then MS/MS scanning is carried out, wherein the scanning range is m/z 100-1500, and the accumulation time is 0.05 s.
The mobile phase was transported using a Waters Nano acquisition UPLC system. Mobile phase a was an aqueous solution containing 0.1% formic acid and mobile phase B was an aqueous solution containing 0.1% formic acid and 98% acetonitrile. The gradient elution procedure was as follows: 0-1min, 4% B; 1-56min, 4% -18% B; 56-70min, 18% -40% B; 70-70.5min, 40% -80% B; 70.5-75.5min, 80% B; 75.5-76min, 80% -4% B; 76-80min, 4% B, flow rate 5 uL/min.
(3) Data retrieval
The wiff files collected from Xcalibur were converted to MGF format using Thermo protein discover meter dam (v1.4) AB _ SCIEX _ MS _ Data _ Converter, and then retrieved in the human (human, protein number 20185) protein database (http:// www.uniprot.org /) using Mascot 2.5.1 software. The library search parameters are as follows: the restriction, maximum leaky cut and fixed modification were not set, and the variable modification was set to oxidation of methionine (+15.9949 Da). The mass tolerance deviation of the parent ion was 50ppm and the fragment ion was 0.2 Da. Peptide fragments derived from Score >20 were analyzed as valid data by controlling false positive rate (FDR) < 1%. The label-free search was performed in human pools using maxquant software, with no restriction, maximum leaky cut and fixed modifications, and the variable modification set to methionine oxidation (+15.9949 Da).
(4) LC-MS/MS obtains target peptide fragment information
More than one thousand pieces of peptidyl fragment information (specific peptidyl fragment information is exemplified in supplementary table 1) were identified in human milk endogenous peptide samples, of which more than about 50% are derived from beta-casein and about 2% are derived from lactoferrin. Polypeptide YEKYLGPQYV was identified in many of the human milk endogenous peptide samples analyzed, indicating its stable presence in human milk. The polypeptide YEKYLGPQYV protein precursor derived from human milk endogenous source is human lactoferrin (f97-114), the isoelectric point of a peptide segment is 6.00, the molecular mass is 1259.42Da, and the polypeptide YEKYLGPQYV protein precursor has 10 amino acids, a shorter sequence and lower relative molecular mass. Obtaining SEQ ID NO: 1, the peptide fragment has an instability coefficient of 13.18, and the aliphatic amino acid index and the hydrophilicity are 68.00 and-0.880, respectively.
Information of SEQ ID No.1
(a) Sequence characterization
Length: 10 amino acid
Type: amino acids
Chain type: single strand
(b) Molecular type: protein
Description of the sequence:
SEQ ID No.1
YEKYLGPQYV
EXAMPLE 2 antibacterial Activity of the Polypeptides
The peptide YEKYLGPQYV was synthesized by Shanghai Jie peptide Biotech Co., Ltd by a solid phase method, and the purity was 96.17%. Gram-positive bacteria escherichia coli and gram-negative bacteria staphylococcus aureus are taken as target strains, and the antibacterial activity of the polypeptide YEKYLGPQYV is investigated.
(1) Strain and resuscitation
Escherichia coli (Escherichia coli K12) and Staphylococcus aureus (Staphylococcus aureus) are preserved at-80 deg.C by glycerol preservation, and are inoculated into liquid LB culture medium and TSB culture medium respectively before experiment, and cultured at 37 deg.C for 12h to recover the strains, and the above recovery operation is repeated for 2-3 times to recover the strains.
(2) Culture medium
Circular petri dishes with a diameter of 90mm were used for the antibacterial experiments. The lower layer of the double-layer culture medium is supported by 15mL of 2% (m/V) agar aqueous solution, 5mL of LB culture medium and TSB culture medium containing 0.7% (m/V) agar and having a bacterial concentration of 1 × 104CFU/mL are respectively added to the upper layer, and the prepared two culture media are sealed and stored at 4 ℃.
(3) Antibacterial experiments
Holes with a diameter of 2 μm were made in the upper layer of the plate, with a spacing of about 3.5 cm. The synthesized standard peptide is divided into 5 mg/piece, 50 mu L of sterile water is added into each piece, 35 mu L of standard peptide aqueous solution is added into each hole after the standard peptide is completely dissolved, and the actual amount of the standard peptide added into each hole is 3.5 mg. And (3) taking water as a blank control, keeping the sample adding volume and the standard peptide sample at 35 mu L, standing the sample added flat plate in a refrigerator at 4 ℃ for 3h, taking out the sample after the sample solution in the hole is completely absorbed and diffused, carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 12h, observing whether a bacteriostatic circle appears, measuring the size of the bacteriostatic circle and recording.
(4) Results of the experiment
The diameters of the inhibition zones of escherichia coli and staphylococcus aureus are shown in table 1 and figure 1, the diameters of the inhibition zones of target peptide fragments are listed in the table, and the result shows that the polypeptide YEKYLGPQYV has inhibitory activity to both gram-negative bacteria and gram-positive bacteria, and the growth inhibitory activity to staphylococcus aureus is slightly higher than that of escherichia coli.
TABLE 1 bacteriostatic results for polypeptide YEKYLGPQYV
"-" indicates no bacteriostatic activity.
Supplementing the peptide fragment information identified in Table 1
Sequence listing
<110> institute of chemistry and physics, large connection of Chinese academy of sciences
<120> human milk endogenous antibacterial polypeptide and application thereof in preparation of anti-inflammatory drugs
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val
1 5 10
Claims (3)
1. The application of the human milk endogenous antibacterial polypeptide in preparing a growth inhibitor of escherichia coli and staphylococcus aureus or a medicine for resisting gastrointestinal infection caused by the escherichia coli and the staphylococcus aureus is characterized in that: the polypeptide is YEKYLGPQYV, and the amino acid sequence of the polypeptide is SEQ ID NO: 1 is shown.
2. Use according to claim 1, characterized in that: the growth inhibitor or the anti-infective medicament takes the polypeptide YEKYLGPQYV as an active ingredient, and a pharmaceutically acceptable carrier or auxiliary material is added or not added.
3. Use according to claim 1, characterized in that: the application of the polypeptide in preparing medicines and/or health products or infant formula foods for preventing and/or reducing intestinal infection diseases caused by escherichia coli and staphylococcus aureus.
Priority Applications (1)
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| WO2007106951A1 (en) * | 2006-03-23 | 2007-09-27 | Agriculture Victoria Services Pty Limited | Antimicrobial protein |
| WO2014060610A2 (en) * | 2012-10-19 | 2014-04-24 | Katholieke Universiteit Leuven, K.U. Leuven R&D | Antimicrobial peptides |
| CN107022001A (en) * | 2017-05-12 | 2017-08-08 | 南京市妇幼保健院 | A kind of breast milk endogenous antibacterial polypeptide of separation and its application |
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| WO2007106951A1 (en) * | 2006-03-23 | 2007-09-27 | Agriculture Victoria Services Pty Limited | Antimicrobial protein |
| WO2014060610A2 (en) * | 2012-10-19 | 2014-04-24 | Katholieke Universiteit Leuven, K.U. Leuven R&D | Antimicrobial peptides |
| CN107022001A (en) * | 2017-05-12 | 2017-08-08 | 南京市妇幼保健院 | A kind of breast milk endogenous antibacterial polypeptide of separation and its application |
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