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Human lactoferrin-like peptide and its application

A technology of lactoferrin and ferritin, which is applied in the field of preparation of human lactoferrin, can solve the problems of low yield, high cost, complicated preparation process, etc.

Active Publication Date: 2022-05-03
GUANGZHOU BIOPHARMACEUTICAL R&D CENT OF JINAN UNIV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, lactoferrin peptides are mainly obtained by hydrolyzing lactoferrin obtained by chromatography, ultrafiltration, salting out, acid precipitation, etc., and then centrifuging, cationic or hydrophobic affinity chromatography, desalting, and freeze-drying. The preparation process is complicated, the yield is low, and the cost is high

Method used

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  • Human lactoferrin-like peptide and its application
  • Human lactoferrin-like peptide and its application
  • Human lactoferrin-like peptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Structural design of human lactoferrin-like peptides

[0022] The human lactoferrin peptide LfcinH is formed by protease digestion of the N-terminal α-helix region 14-31 of human lactoferrin (TKCFQWQRNMRKVRGPPVSCIKR), and a hairpin structure with an amphipathic conformation is formed by an intrachain disulfide bond, and the hydrophobic core is " T-shaped arrangement. Bellamy et al found that bovine lactoferrin peptide LfcinB (FKCRRWQWRMKKLGAPSITCVRR) has antibacterial and bactericidal effects compared with human lactoferrin peptide LfcinH, the former is more than 400 times that of the latter; analysis of its structure found that LfcinB has more positive net charges and more Strong hydrophobic strength, the number of positive charges and the strength of hydrophobicity determine its antibacterial ability, but this characteristic is restrictive. The increase in net charge can only improve its antibacterial activity within a certain range. The same is true for th...

Embodiment 2

[0027] Example 2: Recombinant expression of lactoferrin-like peptide

[0028] The sumo fusion expression system was used for E. coli expression or yeast secretion expression, and the expression plasmids encoding the fusion sumo tag DNA sequence fragments SD1, SD2, SD3, SD4, SD5, SD6, SD7 (SD1, SD2, SD3, SD4, The sequences of SD5, SD6, and SD7 are respectively shown in SEQ ID NO.8-SEQ ID NO.14), heat-shock transformation of E. coli expression bacteria BL21 or electric shock transformation of yeast GS115, resistance screening of positive transformants, respectively with IPTG or methanol High-expression strains were screened for induced expression, and monomeric proteins S1-S7 were obtained after two-step Ni column purification and sumo digestion. S1-S7 fusion sumo gene for recombinant secreted expression such as figure 2 shown.

Embodiment 3

[0029] Embodiment 3: Selective effect on skin epidermal microbial flora

[0030] In this example, human lactoferrin peptide S1 purified in Example 2 and its 6 improved peptides (S2-S7) were used to determine the effect on skin epidermal microbial flora dominant flora Staphylococcus epidermidis (Se ), Propionibacterium acnes (PA) and Pseudomonas (Pse), the minimum inhibitory concentration of the subdominant flora Lactobacillus (La), Staphylococcus aureus (Sa).

[0031] The specific steps are as follows: Dilute the above-mentioned series of bacterial solutions cultivated to the logarithmic growth phase to 2×105~7×105cfu.ml -1 ; Use PBS (sodium phosphate buffer solution, pH 7.0) to prepare human lactoferrin peptide S1 and its 6 improved peptides (S2-S7) as a stock solution of 10 times the highest concentration to be tested, and then perform gradient dilution to obtain a series of Diluted lactoferrin peptide solution; add 2ml of diluted bacterial suspension to the sterilized test...

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Abstract

The invention discloses a class of human lactoferrin peptide, the molecular structure of which is a soluble polypeptide Lfcin structural analog of the 14-31 residue in the N-terminal α-helix region of human lactoferrin, which retains the same folding structure as Lfcin. The human lactoferrin peptide is a polypeptide with a length of no more than 35 amino acids, and its amino acid sequence includes the amino acid sequence of (1) or (2). Lactoferrin-like peptides are similar to those derived from the N-terminal α-helix region of human lactoferrin or the N-terminal α-helix region of bovine lactoferrin. A series of similar structures are designed through structural simulation, and the peptide product is obtained by genetic engineering. Test The synthetic peptide product has significant inhibitory activity against pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas and Propionibacterium acnes in a lower concentration range, but does not affect the normal growth of probiotics such as Lactobacillus , has a regulating effect on skin microecology. It can be applied to general health, such as cosmetics and food.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a preparation method and application of human lactoferrin. Background technique [0002] Lactoferrin peptide (Lactoferricin, Lfcin) is a short peptide with 21-45 amino acid residues released from the N-terminus of lactoferrin after being digested by gastric juice. Its function has all the biological functions of lactoferrin except that it cannot bind iron ions. active. Among the many physiological functions of lactoferrin peptide, the most striking is its antibacterial effect, which has a broad spectrum of antibacterial effects, including many Gram-negative and positive pathogenic bacteria and fungi, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus , Streptococcus and Candida albicans, etc. At present, there are two main views on the antibacterial mechanism of lactoferrin peptide: one is to block the utilization of iron by bacteria, and the other is to bind pos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/79C12N15/12C12N15/62A61K38/40A61P31/04
CPCC07K14/79A61P31/04C07K2319/35A61K38/00
Inventor 项琪黄亚东肖雪倪佩红
Owner GUANGZHOU BIOPHARMACEUTICAL R&D CENT OF JINAN UNIV CO LTD