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Antibacterial peptide separated from cow blood and code sequence and application thereof

An antibacterial peptide and an isolated technology are applied in the field of antibacterial peptides and their coding sequences, which can solve the problems such as research reports on the isolation and identification of antibacterial peptides that have not yet been seen, and achieve safe accumulation of poisoning, no toxic side effects, convenient artificial synthesis, and simple structure. Effect

Inactive Publication Date: 2009-12-23
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there is no research report on the isolation and identification of antimicrobial peptides from cow blood hemoglobin

Method used

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  • Antibacterial peptide separated from cow blood and code sequence and application thereof
  • Antibacterial peptide separated from cow blood and code sequence and application thereof
  • Antibacterial peptide separated from cow blood and code sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Collection and processing of blood

[0029]After the slaughter of healthy dairy cows, collect their blood in a test tube containing heparin (15IU / ml) by aseptic method, place it in an ice bath, send it to the laboratory within 1 hour, and wash it with 0.9% sterile normal saline immediately red blood cells 3 times, and then lysed red blood cells with 0.83% ammonium chloride solution (volume ratio 3:1). After centrifuging at 700×g at 4° C. for 15 minutes to remove white blood cells, the solution containing the heme polypeptide was lyophilized and dissolved in 0.01% acetic acid.

Embodiment 2

[0030] Embodiment 2: the purification of antibacterial peptide

[0031] The antimicrobial peptides in the crude heme solution were separated by ion exchange column chromatography (reversible adsorption of cationic peptides and ion exchange columns immobilized by negative ions). Pass the crude sample through an ion exchange column. The cationic complex is bound to the anionic resin, the non-cationic molecules are washed away by 25mM ammonium acetate, and the cationic molecules are eluted by 10% acetic acid. The collected solution was poured into the column at a rate of 20 mL / h, and its absorbance was monitored at a wavelength of 230 nm with a double-beam UV-Vis spectrophotometer. The eluted peaks were collected (5 mL / peak), lyophilized, and resuspended with 0.01% acetic acid. After separation, there are 4 peaks in total, E1 is an anion peak, E2, E3 and E4 are cation peaks, see figure 1 , through antibacterial activity detection (the method of embodiment 3), find that peak li...

Embodiment 3

[0033] Embodiment 3: detection of antibacterial activity

[0034] Antimicrobial activities of peptides separated by ion exchange and reversed-phase high-performance liquid chromatography (tested strains were Staphylococcus aureus NCTC 4163, Escherichia coli O111 and Candida albicans 3135A) were determined by plate diffusion method.

[0035] Spread the above-mentioned 3 kinds of tested microorganisms on the sterilized lower layer agar medium containing nutrients (10mL trypticase casein soybean broth (product of Cole-Parmer Company), 10g ultrapure agar (Sigma A-6013 ), 1L of distilled water, pH 7.4), punch holes with a hole diameter of about 2 mm, heat the alcohol lamp slightly to seal the back, add 5 μL of test samples with a concentration of 0.8 μg / ml to each well with a pipette, and Each plate includes positive and negative controls. Positive controls used common antibiotics: polymyxin B against gram-negative bacteria, nisin (nisin) against gram-positive bacteria, and nystat...

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Abstract

The invention relates to an antibacterial peptide with antibacterial activity separated from cow blood, a code sequence and an application thereof. The antibacterial peptide has 1 to 25 amino acid sequences in a sequence table SEQ ID NO: 1.Except for the common property of the common antibacterial peptide such as wide antibacterial spectrum, quick sterilization, high selectivity, no drug resistance generation and no teratogenic action, the antibacterial peptide also has the flowing advantages that the structure is simple; the artificial synthesis is convenient; no antigenicity exists when the antibacterial peptide is applied to mammals; the antibacterial peptide is separated from the red blood cell of the cow blood; and the red blood cell is one of animal blood components, is involved in the process of blood circulation, and has no hemolytic toxicity or low hemolysis rate. The antibacterial peptide not only can be used for preparing a medicament for treating gram positive bacteria, gram negative bacteria, and epiphyte or viral infection, but also can be applied to the fields such as milk fresh-keeping, or other food preservatives, feed mould inhibitors and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial peptide with antibacterial activity isolated from cow blood, its coding sequence and application. Background technique [0002] Antimicrobial peptides (antimicrobial peptides) are a class of polypeptides with antibacterial activity produced by biological cell genes. They are natural immune active molecules produced by the body in the process of biological evolution to adapt to the environment and survive themselves, and are important media of natural immunity. The molecular weight of antimicrobial peptides is small, and the peptide chain is generally 10 to 50 amino acid residues. Antimicrobial peptides have been found not only to have broad-spectrum antibacterial (gram-positive and gram-negative) activity, but also to fungi (such as Candida albicans, etc.), viruses (such as influenza virus, HIV, etc.), parasites (such as Nematodes, flagellates, etc.) and...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/18C07K1/14A61K38/17A61P31/00A23L1/305A23K1/17A23K20/195A23L33/18
Inventor 胡建和刘兴友赵坤王三虎王自良杭柏林陈俊杰王丽荣杨理王青
Owner HENAN INST OF SCI & TECH
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