CN112500475B - Human lactoferrin-like peptide and application thereof - Google Patents
Human lactoferrin-like peptide and application thereof Download PDFInfo
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Abstract
The invention discloses a human lactoferrin-like peptide, the molecular structure of which is a soluble polypeptide Lfcin structural analogue of human lactoferrin N-terminal alpha-helical region residues 14-31, a folding structure which is the same as that of Lfcin is reserved, the human lactoferrin-like peptide is a polypeptide with the length of not more than 35 amino acids, and the amino acid sequence of the human lactoferrin-like peptide comprises the amino acid sequence containing (1) or (2). The lactoferrin-like peptide is similar to a structure of an N-terminal alpha-helical region derived from human lactoferrin or an N-terminal alpha-helical region derived from bovine lactoferrin, a series of similar structures are designed through structural simulation, a polypeptide product is obtained through genetic engineering, and the synthesized polypeptide product is tested to have remarkable inhibitory activity on pathogenic bacteria such as escherichia coli, staphylococcus aureus, staphylococcus epidermidis, pseudomonas and propionibacterium acnes in a lower concentration range, but the normal growth of probiotics such as lactobacillus is not influenced, so that the lactoferrin-like peptide has a regulating effect on skin micro-ecology. Can be used in the fields of health promotion, such as cosmetics and food.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to a preparation method and application of human lactoferrin-like protein.
Background
Lactoferricin (Lfcin) is a short peptide with 21-45 amino acid residues released from the N-terminal of lactoferrin after digestion in gastric juice, and has the function of possessing all the biological activities of lactoferrin except that the lactoferrin cannot be bound with iron ions. Among the physiological functions of lactoferricin, the most remarkable one is its antibacterial action, which is broad-spectrum and includes many gram-negative and gram-positive pathogenic bacteria and fungi, such as escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, streptococcus, candida albicans, etc., and there are two main points of the current antibacterial mechanism for lactoferricin: one is to block the utilization of iron by bacteria, and the other is to form perforations by the interaction of positively charged lactoferricin and phospholipid groups on the bacterial membrane, which leads to the leakage of bacterial contents and cell death. Lactoferrin peptides also inhibit iron-induced lipid peroxidation and reduce the production of oxygen radicals. The lactoferrin peptide also has an immunoregulation function, and is a hot spot for research and development because various biological functions of the lactoferrin peptide are widely concerned. But the regulation function of the skin microbial ecological agent on the skin microbial ecological agent is not reported. At present, lactoferrin is mainly prepared by hydrolysis through methods such as chromatography, ultrafiltration, salting out, acid precipitation and the like, and is prepared by centrifugation, cation or hydrophobic affinity chromatography, desalination and freeze drying.
The existing research shows that the bovine lactoferrin peptide has stronger antibacterial activity than that of human lactoferrin peptide, the former is more than 400 times of that of the latter, the sequence structures of the bovine lactoferrin peptide and the human lactoferrin peptide are analyzed, structural simulation and design are carried out based on the optimal hydrophobic strength and net charge number range, a series of novel improved peptides based on the secondary structure of the human lactoferrin peptide are designed, a series of human lactoferrin peptide is obtained by combining genetic engineering recombination expression with a two-step chromatography, harmful bacteria which are not beneficial to skin integrity can be selectively inhibited, probiotics are nourished, and the skin microecological balance is maintained.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a series of human lactoferrin-like peptides and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a human lactoferrin-like peptide, the molecular structure of which is a soluble polypeptide Lfcin structural analogue of human lactoferrin N-terminal alpha-helical region residues 14-31, with the same folding structure as Lfcin preserved, the human lactoferrin-like peptide being a polypeptide with a length of not more than 35 amino acids, the amino acid sequence of which comprises the amino acid sequence comprising (1) or (2):
(1) amino acid sequences SEQ ID NO.1-SEQ ID NO.7 shown in soluble polypeptide containing 14-31 residues of the N-terminal alpha-helical region of the human lactoferrin-like protein;
(2) the amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.7 is substituted, deleted or added with one or more than one and has the amino acid sequence with the same function as the amino acid sequence shown in SEQ ID NO.1-SEQ ID NO. 7.
The preferable amino acid sequence of the human lactoferrin-like peptide is shown in SEQ ID NO.1-SEQ ID NO.7, and the coded nucleotide sequence is shown in NO.8-SEQ ID NO. 14.
The expression vector containing the nucleotide sequence is host vector of colibacillus, yeast, bacillus, insect or Chinese hamster ovary cell.
Compared with the prior art, the invention has the following beneficial effects: the lactoferrin-like peptide is similar to a structure derived from a human lactoferrin N-terminal alpha-helical region (45 amino acid residues) or a bovine lactoferrin N-terminal alpha-helical region (23 amino acid residues), a series of similar structures are designed through structural simulation, a polypeptide product is obtained by utilizing a genetic engineering recombinant expression or polypeptide synthesis method, and the synthesized polypeptide product is tested to be in a lower concentration range (5-25 mu mol-1) Has obvious inhibiting activity on pathogenic bacteria such as escherichia coli, staphylococcus aureus, staphylococcus epidermidis, pseudomonas, propionibacterium acnes and the like, does not influence the normal growth of probiotics such as lactobacillus and the like, and has regulating effect on skin micro-ecology. The screened polypeptide product can be applied to the fields of large health, such as cosmetics, foods and the like.
Drawings
FIG. 1 is a spatial structure diagram of simulation S1-S7 by Phyre2 software.
FIG. 2 is a diagram of recombinant secretion expression of the S1-S7 fusion sumo gene.
Lane1 and 6 were uninduced extracellular, 2 was SD1, 3 was SD2, 4 was SD3, 5 was SD4, 7 was SD5, 8 was SD6, and 9 was SD7 for 72 hours of extracellular induction.
FIG. 3 shows the working concentration (5. mu. mol. L) of human lactoferricin S1 and its modified peptide S2-S7-1) Effect on the growth of Staphylococcus epidermidis (Se), Propionibacterium Acnes (PA), Pseudomonas (Pse), Staphylococcus aureus (Sa) and Lactobacillus (La) respectively under the conditions.
FIG. 4 shows the inhibition of growth of acne vulgaris by lactoferricin observed by transmission electron microscopy;
a is a control group; b is the S2 treatment group.
FIG. 5 is a patch test, wherein A is PBS, B is blank, C is a sample of stock solution of human lactoferrin peptide S2 of example 2, and D is C sample control buffer salt.
FIG. 6 is a VISA facial picture of a subject before and after four weeks using the human lactoferrin peptide S2 of example 2; wherein, A is before use, and B is after four weeks of use.
Fig. 7 shows statistics of changes of parameters such as red pigment, lipid content, and purple on the front and back of human lactoferrin peptide S2 in example 2, before use, W0, and after use, W4.
The specific implementation mode is as follows:
example 1: structure design of human lactoferrin-like peptide
The human lactoferrin peptide LfcinH is formed by digesting the N-terminal alpha helical region 14-31 of human lactoferrin by protease (TKCFQWQRNMRKVRGPPVSCIKR), and a hairpin structure with an amphiphilic conformation is formed by an intrachain disulfide bond, and a hydrophobic core is arranged in a T shape. The research of Bellamy et al finds that the bacteriostatic and bactericidal effects of bovine lactoferrin peptide LfcinB (FKCRRWQWRMKKLGAPITCVRR) are 400 times of those of human lactoferrin peptide LfcinH; the structure of the LfcinB is analyzed to find that the LfcinB has more positive charge net charge number and stronger hydrophobic strength, the positive charge number and the hydrophobic strength determine the antibacterial capability of the LfcinB, but the characteristic has limitation, the net charge increase only improves the antibacterial activity of the LfcinB within a certain range, the antibacterial peptide has the same hydrophobicity, the hydrophobicity is moderate, the LfcinB can be dissolved in water and rapidly transported to target microorganisms (low hydrophobicity is required) and can also act with a hydrophobic region of a cytoplasmic membrane bilayer (high hydrophobicity is required), therefore, in the optimal design of the LfcinH derivative peptide structure, an optimal range of the hydrophobic strength and the net charge number exists, and in the range, the hydrophobicity and the positive charge number are positively correlated with the antibacterial activity. Simulating the spatial structure of the peptide by using the Phyre2 software; calculating a polypeptide hydrophilic value (GRAVY value) and a net positive charge number by using a protein hydrophobicity analysis tool and DNAstar software, predicting the antibacterial activity of the derived peptides by using a prediction tool of an APD antibacterial peptide database and a CAMP antibacterial peptide database to obtain the optimal LfcinH derived peptides S2-S7, wherein the amino acid sequences of the LfcinH derived peptides are respectively shown as SEQ ID NO.1-SEQ ID NO. 7.
The sequence information of the S1-S7 polypeptide is designed as shown in Table 1:
TABLE 1
Example 2: recombinant expression of lactoferricin-like peptides
Coli expression or yeast secretion expression is carried out by using a sumo fusion expression system, expression plasmids (the sequences of SD1, SD2, SD3, SD4, SD5, SD6 and SD7 are respectively shown in SEQ ID NO.8-SEQ ID NO. 14) of fusion sumo label DNA sequence fragments SD1, SD2, SD3, SD4, SD5, SD6 and SD7 are synthesized by agilawood, a large intestine expression bacterium BL21 or an electric shock transformed yeast GS115 is subjected to heat shock transformation, a positive transformant is subjected to resistance screening, high expression strains are subjected to induction expression screening by using IPTG or methanol respectively, and a monomeric protein S1-S7 is obtained through two steps of Ni column purification and sumo enzyme digestion. The recombinant secretory expression of the S1-S7 fusion sumo gene is shown in FIG. 2.
Example 3: selective action on skin epidermal microbial flora
In this example, the minimum inhibitory concentrations of the human lactoferrin peptide S1 and 6 modified peptides thereof (S2-S7) purified in example 2 to staphylococcus epidermidis, Propionibacterium Acnes (PA) and pseudomonas species (Pse), lactobacillus species (La) and staphylococcus aureus (Sa), which are dominant flora, of the epidermal microflora of skin, were measured by broth dilution method.
The method comprises the following specific steps: diluting the series of bacterial liquid cultured to logarithmic growth phase to 2 × 105-7 × 105cfu-1(ii) a Preparing human lactoferrin peptide S1 and 6 modified peptides (S2-S7) thereof into stock solution with the highest concentration to be measured being 10 times by using PBS (sodium phosphate buffer solution, pH value being 7.0), and then carrying out gradient dilution to obtain lactoferrin peptide solution with serial dilution concentration; respectively adding 2ml of diluted bacterial suspension into a sterilized test tube, respectively adding 200 mul of lactoferrin to be detected with corresponding concentration, taking a mixed system of a sterile liquid culture medium and water as a blank, taking ampicillin as a positive control, and taking pure water as a negative control. The results were observed after incubation for 24 hours in a 37-degree or 30-degree shaking incubator, and the minimum lactoferricin concentration that could prevent bacterial growth was taken as the MIC.
The results are shown in Table 2:
TABLE 2 Lactoferrin peptide minimum inhibitory concentration in vitro (MIC. mu. mol.L)-1)
| Name(s) | Se | PA | Pse | Sa | La |
| S1 | >250 | 40 | >250 | >250 | >250 |
| |
5 | 5 | 5 | 150 | >250 |
| |
20 | 20～40 | 20 | 200～250 | >250 |
| S4 | 20 | 100 | 20 | >250 | >250 |
| S5 | 20 | 40 | >250 | >250 | >250 |
| S6 | 20 | 5～20 | 5 | >250 | >250 |
| S7 | >250 | >250 | >250 | >250 | >250 |
From the experimental results of example 3, table 2 shows: the human lactoferrin peptide S1 and the modified peptides S2-S6 respectively have different degrees of inhibitory action on pathogenic bacteria such as staphylococcus epidermidis (Se), Propionibacterium Acnes (PA), pseudomonas (Pse) and staphylococcus aureus (Sa), the inhibitory action is reduced along with the reduction of the positive charge number of the modified peptide, the antibacterial action is lost when the positive charge number is less than 6, for example, S7 loses the antibacterial activity at a higher concentration of 250 mu mol-1The inhibitory effects on Staphylococcus epidermidis (Se), Propionibacterium Acnes (PA), Pseudomonas (Pse) and Staphylococcus aureus (Sa) were not evident below. The modified peptide S2 has the strongest bacteriostatic action, and the MIC values of staphylococcus epidermidis (Se), Propionibacterium Acnes (PA), pseudomonas (Pse) and staphylococcus aureus (Sa) are respectively 5, 5 and 150 mu mol-1. The human lactoferrin peptide S1 and the modified peptide S2-S6 have no influence on the growth of the probiotic lactobacillus (La), and can promote the growth of the lactobacillus (La) to a certain extent to play a nutritional role (shown in the picture of fig. 3 La).
Example 4: mechanism of inhibition of acne bacteria by lactoferricin
The human lactoferrin peptide-like S2 having the strongest effect on Propionibacterium acnes prepared in example 2 was formulated to a concentration of 10. mu.g.ml-1Sampling and fixing propionibacterium acnes cultured in logarithmic phase after 4 hours of action, and observing by transmission electron microscope slide, wherein the result is shown in figure 4, and the growth inhibition effect of human lactoferrin peptide S2 on propionibacterium acnes is mainly due to bacterial cell wall and bacterial cell wallThe cell membrane causes perforation, resulting in massive solute leakage and massive bacterial death.
Example 5: stimulation of skin cells by human lactoferrin-like peptides
(1) Cytotoxicity of human lactoferrin-like peptides on epidermal cells in vitro
The cytotoxicity of lactoferrin on 3T3 and Hacat cells within a certain action concentration range was determined according to MTT method, a biological activity assay method in the third division of the pharmacopoeia of China 2015 edition.
TABLE 3 cytotoxicity of lactoferricin on skin cells
The results show that: lactoferrin S1 and its 6 modified peptides at working concentration (5-20. mu. mol.L)-1) Is substantially non-irritating to skin cells under conditions.
(2) Stimulating effect of lactoferricin on normal skin
Human lactoferrin-like peptides were tested for their stimulation of normal skin by the human skin patch test.
18 volunteers aged 20-45 years, with healthy skin and no history of skin allergy, were selected, and were randomly divided into 2 groups for patch experiments, one sample was used for each group, in accordance with the volunteer selection criteria. A qualified patch device is selected, 50 mul of the similar human lactoferrin S2(5 mu g/ml) protein liquid and a control solvent (50mM phosphate (pH 7.0)) in example 2 are uniformly smeared in the patch device by a closed patch experimental method, a special adhesive tape is externally applied to the back of a subject, the subject is removed after 24h, the skin reaction is observed at 0 th, 0.5 th, 24 th and 48h after the removal, and the result is recorded according to the skin reaction grading standard in the cosmetic hygiene standard, as shown in figure 5.
The result of the human body skin patch experiment shows that: the skin reactions of the 18 subjects were observed at hours 0, 0.5, 24, 48, of which 0 had adverse skin reactions, indicating that the protein stock solution containing the human lactoferrin-like peptide was safe and non-irritating.
Example 6: application of lactoferricin on skin
30 test subjects with body health meeting the conditions are selected, 5ug/ml of stock solution of human lactoferrin-like peptide S2 is smeared for 2 times every day, the anti-acne effect of the subjects after four weeks (W4) is observed by using VISIA facial images, and the purple characteristic of the facial skin is detected by a CK skin tester, and the results are shown in fig. 6 and 7. After 4 weeks of using the stock solution of the human lactoferrin peptide S2, the value of facial acne red pigment of 30 vaccinia muscle volunteers is reduced by 39.8 percent compared with that before the stock solution (W0), the oil secretion is reduced by 41.9 percent, the extremely significant difference (p is less than 0.01) exists, the facial purple characteristic count is reduced by 50.8 percent compared with that before the stock solution is used, the extremely significant difference (p is less than 0.01) exists, the porphyrin on the face of the testee is obviously reduced, the porphyrin is a metabolite of bacteria and can block pores to cause acne, the stock solution of the human lactoferrin peptide S2 can inhibit the growth of facial pathogenic bacteria, regulate the microecology of the facial skin and effectively improve the acne.
Sequence listing
<110> medical biotechnology research and development center of university of Guangzhou river, Guangzhou peptidergic Gene bioengineering, Inc
<120> human lactoferrin peptide and application thereof
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catcatcatc atcatcatgg tatgtctgat tctgaagtta accaagaagc taagccagaa 60
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catcatcatc atcatcatgg tatgtctgat tctgaagtta accaagaagc taagccagaa 60
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gaaatttttt ttaagattaa gaagactact ccacttagaa gacttatgga agcttttgct 180
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catcatcatc atcatcatgg tatgtctgat tctgaagtta accaagaagc taagccagaa 60
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gaaatttttt ttaagattaa gaagactact ccacttagaa gacttatgga agcttttgct 180
aagagacaag gtaaggaaat ggattctctt agatttcttt acgatggtat tagaattcaa 240
gctgatcaaa ctccagaaga tcttgatatg gaagataacg atattattga agctcataga 300
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catcatcatc atcatcatgg tatgtctgat tctgaagtta accaagaagc taagccagaa 60
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gctgatcaaa ctccagaaga tcttgatatg gaagataacg atattattga agctcataga 300
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catcatcatc atcatcatgg tatgtctgat tctgaagtta accaagaagc taagccagaa 60
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gaaatttttt ttaagattaa gaagactact ccacttagaa gacttatgga agcttttgct 180
aagagacaag gtaaggaaat ggattctctt agatttcttt acgatggtat tagaattcaa 240
gctgatcaaa ctccagaaga tcttgatatg gaagataacg atattattga agctcataga 300
gaacaaattg gtggtactaa gtgttttcaa tggcaaagat ggatgagaaa ggttagatgg 360
ccaccagttt cttgtatt 378
Claims (5)
1. A human lactoferrin-like peptide has a molecular structure of a soluble polypeptide Lfcin structural analogue of human lactoferrin N-terminal alpha-helical region residues 14-31, and a folding structure identical to that of Lfcin is retained, wherein the human lactoferrin-like peptide is a polypeptide with the length of not more than 35 amino acids, and the amino acid sequence of the human lactoferrin-like peptide is shown in SEQ ID NO. 2-SEQ ID NO. 6.
2. Nucleic acid encoding the human lactoferrin peptide-like composition of claim 1, wherein the sequence of the nucleic acid is shown in SEQ ID No.9 to SEQ ID No. 13.
3. A fusion sumo tag DNA is characterized in that the sequence of the DNA is shown in SEQ ID NO. 16-SEQ ID NO. 20.
4. A host cell comprising the nucleic acid of claim 2, wherein the host cell is Escherichia coli, yeast, Bacillus, insect cell or Chinese hamster ovary cell.
5. Use of the human lactoferrin peptide of claim 1 in the manufacture of a medicament for inhibiting propionibacterium acnes or staphylococcus aureus.
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| CN101687017A (en) * | 2007-05-14 | 2010-03-31 | 方塔拉合作集团有限公司 | Methods of immune or hematological enhancement, inhibiting tumour formation or growth, and treating or preventing cancer, cancer symptoms, or the symptoms of cancer treatments |
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Non-Patent Citations (2)
| Title |
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| Human Lactoferrin and Peptides Derived from a Surface-Exposed Helical Region Reduce Experimental Escherichia coli Urinary Tract Infection in Mice;LILIANA A. HÅVERSEN;《INFECTION AND IMMUNITY》;20001031;第68卷(第10期);第5816–5823页 * |
| 人乳铁蛋白肽的分子改良及在大肠杆菌中的表达研究;蔡屾;《化学与生物工程》;20111031;第28卷(第10期);第30页表1 * |
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