阅读说明：本技术 类人乳铁蛋白肽及其应用 (Human lactoferrin-like peptide and application thereof ) 是由 项琪 黄亚东 肖雪 倪佩红 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种类人乳铁蛋白肽,其分子结构为人乳铁蛋白N端α-螺旋区14-31残基的可溶性多肽Lfcin结构类似物,保留有与Lfcin相同的折叠结构,所述类人乳铁蛋白肽为具有不大于35个氨基酸长度的多肽,其氨基酸序列包括含有(1)或(2)的氨基酸序列。类乳铁蛋白肽与来源于人乳铁蛋白N-末端α-螺旋区或牛乳铁蛋白N-末端α-螺旋区结构类似,通过结构模拟设计系列类似结构物,利用基因工程获取多肽产物,测试合成的多肽产物在较低浓度范围内对大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌、假单胞菌和痤疮丙酸杆菌等病原菌具有显著抑制活性,但不影响乳酸杆菌等益生菌的正常生长,对皮肤微生态具有调节作用。可应用于大健康,如化妆品和食品等领域。(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.)

1. A human lactoferrin-like peptide having a molecular structure of a soluble polypeptide Lfcin structural analogue of human lactoferrin N-terminal alpha-helical region residues 14-31, with a folding structure identical to that of Lfcin preserved, the human lactoferrin-like peptide being a polypeptide having a length of not more than 35 amino acids, the amino acid sequence of which comprises an 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.

2. Lactoferrin according to claim 1, characterized in that its amino acid sequence is as shown in SEQ ID No.1-SEQ ID No. 7.

3. A lactoferricin nucleotide sequence as claimed in claim 2 characterised by the sequence shown in No.8-SEQ ID No. 14.

4. A fusion sumo tag DNA sequence is characterized in that the sequence is shown as NO. 15-SEQ ID NO. 21.

5. An expression vector comprising the nucleotide sequence of claim 3, wherein the host vector is an E.coli, yeast, Bacillus, insect or Chinese hamster ovary cell.

6. Use of lactoferricin according to claim 1 in the manufacture of a medicament for inhibiting propionibacterium acnes or escherichia coli or staphylococcus aureus.

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 polypeptide product is tested and synthesizedThe resulting polypeptide product is in a lower concentration range (5-25. mu. mol. L^-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 the simulation S1-S7 of the 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.

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 (FKCRRWFQRMKKLGAPITCVRR) are 400 times of that 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

The method comprises the steps of performing large intestine expression or yeast secretion expression by using a sumo fusion expression system, synthesizing 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 tag DNA sequence fragments SD1, SD2, SD3, SD4, SD5, SD6 and SD7 by agilawood, transforming large intestine expression bacteria BL21 or transforming GS115 by electric shock, screening positive yeast by resistance, inducing expression and screening high expression strains by IPTG or methanol respectively, and obtaining a monomer protein S1-S7 by 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 microbial flora of skin, were determined 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 3:

TABLE 3 Lactoferrin peptide minimum inhibitory concentration in vitro (MIC. mu. mol.L)^-1)

Name (R)	Se	PA	Pse	Sa	La
						S1	>250	40	>250	>250	>250
S2	5	5	5	150	>250
						S3	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 3 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 inhibitory action is lost when the positive charge number is less than 6, for example, S7 is used 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 thereof have no influence on the growth of the probiotic lactobacillus (La) and can promote the lactobacillus (La) to a certain extent) Grow and play a nutritional role (see 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^-1The propionibacterium acnes cultured in the logarithmic phase is taken for 4 hours and then is sampled and fixed, and transmission electron microscope slide production observation is carried out, the result is shown in figure 4, and the growth inhibition effect of the human lactoferrin-like peptide S2 on the propionibacterium acnes is mainly due to perforation on bacterial cell walls and cell membranes, so that a large amount of solutes leak out and the bacteria die in a large amount.

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 4 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 human lactoferrin peptide-like S2 stock solution, the facial acne red pigment value of 30 vaccinia muscle volunteers is reduced by 39.8 percent compared with that before the use (W0), the oil secretion is reduced by 41.9 percent, the very 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 use (p is less than 0.01), the very significant difference (p is less than 0.01) indicates that the porphyrin on the face of a subject is obviously reduced, the porphyrin is a metabolite of bacteria and can block pores to cause acne, and the human lactoferrin peptide-like S2 stock solution can inhibit the growth of facial pathogenic bacteria, regulate the microecology of the facial skin and effectively improve the acne.

Sequence listing

<110> Guangzhou river south university center for research and development of medical biotechnology, Guangzhou peptidergic Gene bioengineering, Inc

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