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Antimicrobial peptide derived from Holstein dairy cow spleen and its preparation method and application

An antibacterial peptide and cow technology, applied in the biological field, can solve the problems such as research reports on the separation and identification of antibacterial peptides that have not yet been seen, and achieve the effects of convenient artificial synthesis, high antibacterial effect, and difficulty in accumulating poisoning.

Active Publication Date: 2020-05-08
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no research report on the isolation and identification of antimicrobial peptides from bovine spleen

Method used

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  • Antimicrobial peptide derived from Holstein dairy cow spleen and its preparation method and application
  • Antimicrobial peptide derived from Holstein dairy cow spleen and its preparation method and application
  • Antimicrobial peptide derived from Holstein dairy cow spleen and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Preparation of spleen crude extract

[0032] Take the spleen of a healthy Holstein cow, remove the surface coating, and cut the spleen into about 3mm 3 Place the small pieces in a sterilized beaker, add an appropriate amount of pre-cooled sterilized physiological saline, mash the tissue, and homogenate; the homogenate is broken twice in an ultrasonic breaker (8s-4s, 35% Amp), 15min / Second-rate. Add an equal volume of 5% acetic acid to the homogenized spleen, stir in an ice bath for 6 hours; then centrifuge at 8000 rpm at 4°C for 30 minutes, and take the supernatant; evaporate the supernatant at 38-40°C for 6 hours -8h, until the acetic acid is completely evaporated; collect the remaining liquid after rotary evaporation, and freeze-dry to obtain the crude extract of spleen.

Embodiment 2

[0033] Embodiment 2: the ion-exchange chromatography purification of antimicrobial peptide

[0034] The spleen crude extract is dissolved with 0.01% acetic acid to obtain a spleen crude extract solution, and the spleen crude extract solution is passed through a cation exchange column, and the cationic components in the spleen crude extract solution are bound to the cation exchange column. Ammonium acetate buffer (pH5.4) passes through the cation exchange column at a flow rate of 50mL / h to wash away unbound components, and then use 10% acetic acid to elute the bound cations on the cation exchange column at a flow rate of 50mL / h Components, during the elution process, measure the absorbance of the elution components at a wavelength of 230nm with a UV detector, and collect the elution peaks (5ml / peak). After separation, there are two elution peaks, peak A is an anion peak and peak B is a cation peak (see figure 1 ). Antibacterial activity detection (see Example 5 for the detect...

Embodiment 3

[0037] Embodiment 3: the reversed-phase high-performance liquid chromatography purification of antimicrobial peptide

[0038] The collected cationic elution peak components (peak B) were further separated by reverse phase high performance liquid chromatography (RP-HPLC). The chromatographic column that described reverse-phase high performance liquid chromatography adopts is the product of U.S. Phenomenex Company, and its model is Jupiter 5uC18 The eluents used are buffer A and buffer B, wherein, the buffer A is: 5% acetonitrile, 0.1% trifluoroacetic acid (the preparation method is: add 5 mL of acetonitrile to 95 mL of deionized water, mix well, and then Add 0.1mL trifluoroacetic acid therein, mix uniformly to obtain buffer solution A); The buffer solution B is: 95% acetonitrile, 0.1% trifluoroacetic acid (the preparation method is: add 5mL deionized water to 95mL acetonitrile, mix and then add 0.1mL trifluoroacetic acid therein, and mix uniformly to obtain buffer B); the elut...

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Abstract

The invention discloses an antibacterial peptide which is obtained by being separated from a Holstein cow spleen and has antibacterial activity, namely a Holstein cow spleen-derived antibacterial peptide, wherein an amino acid sequence of the Holstein cow spleen-derived antibacterial peptide is shown in SEQ ID NO.1. The antibacterial peptide disclosed by the invention is separated from a cow spleen, and the spleen is one of immune organs of an animal and participates blood storage and immunologic processes, so that the antibacterial peptide is extremely low in hemolysis ratio; meanwhile, the antibacterial peptide has some safe biological characteristics without toxic or side effect such as broad antibacterial spectrum, speediness in sterilization, high selectivity, no drug resistance, no teratogenic effect and difficulty in formation of accumulative intoxication, is broad in antibacterial spectrum, has efficient antibacterial effect on Gram negative bacteria, Gram positive bacteria and fungi, can be used for preparing medicines used for treating the Gram negative bacterial, Gram positive bacterial or / and fungal infection and also can be used as a feed additive or a food additive.

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 the spleen of Holstein cows, its coding sequence and application. Background technique [0002] The application of antibiotics has brought great convenience to the treatment of human and animal diseases. However, with the extensive use or abuse of antibiotics, the emergence of multidrug-resistant bacteria has brought great challenges to the treatment of human and animal diseases. At the same time, the problem of antibiotic residues and side effects of antibiotics have posed a huge threat to human and animal health. Therefore, finding alternatives to antibiotics has become an inevitable choice. [0003] Antimicrobial peptides (AMPs) are an important part of the innate immune system of living organisms. Antimicrobial peptides have a small molecular weight and generally consist of 10 to 60 amino acid residu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K1/14C07K1/20C07K1/18A61K38/17A61P31/04A61P31/10A23L33/18A23K20/147
CPCA23K20/147A23L33/18A61K38/00C07K14/4723
Inventor 杭柏林胡建和徐彦召孙亚伟王青王磊夏小静夏一赫
Owner HENAN INST OF SCI & TECH
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