Holstein cow spleen-derived antibacterial peptide as well as preparation method and application thereof

An antimicrobial peptide and spleen technology, applied in the field of antimicrobial peptides and their coding sequences, can solve problems such as research reports on the isolation and identification of antimicrobial peptides that have not yet been seen, and achieve the effect of convenient artificial synthesis, simple structure, and high-efficiency antibacterial effect

Active Publication Date: 2017-10-27
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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  • Holstein cow spleen-derived antibacterial peptide as well as preparation method and application thereof
  • Holstein cow spleen-derived antibacterial peptide as well as preparation method and application thereof
  • Holstein cow spleen-derived antibacterial peptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of crude spleen extract

[0032] Take the spleen of healthy Holstein cows, remove the surface coating, and cut the spleen into about 3mm 3 Place the small pieces in a sterile beaker, add an appropriate amount of pre-cooled sterile physiological saline, mash and homogenize the tissue; crush the homogenate twice with an ultrasonic disintegrator (8s-4s, 35% Amp), 15min / Times. 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 for 30 min at 4°C, and take the supernatant; rotate the supernatant at 38-40°C 6 -8h, until the acetic acid is completely evaporated; collect the remaining liquid after rotary evaporation and freeze-dry to obtain the crude spleen extract.

Embodiment 2

[0033] Example 2: Ion exchange chromatography purification of antibacterial peptides

[0034] Dissolve the crude spleen extract with 0.01% acetic acid to obtain the crude spleen extract solution. Pass the crude spleen extract solution through the cation exchange column. The cation components in the crude spleen extract solution are bound to the cation exchange column. First use 25mmol / L Ammonium acetate buffer (pH 5.4) was passed through the cation exchange column at a flow rate of 50 mL / h to wash away unbound components, and then the bound cations on the cation exchange column were eluted with a concentration of 10% acetic acid at a flow rate of 50 mL / h Components, the elution process uses an ultraviolet detector to measure the absorbance of the eluted component at a wavelength of 230nm, and collect the elution peak (5ml / peak). After separation, there are two elution peaks, peak A is the anion peak, and peak B is the cation peak (see figure 1 ). After antibacterial activity det...

Embodiment 3

[0037] Example 3: Reversed-phase high performance liquid chromatography purification of antibacterial peptides

[0038] The collected cation elution peak components (peak B) were further separated by reversed-phase high performance liquid chromatography (RP-HPLC). The chromatographic column used in the reversed-phase high-performance liquid chromatography is a product of Phenomenex, USA, 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 5mL acetonitrile to 95mL deionized water, mix, and then Add 0.1 mL of trifluoroacetic acid and mix well to obtain buffer A); the buffer B is: 95% acetonitrile, 0.1% trifluoroacetic acid (the preparation method is: add 5 mL of deionized water to 95 mL of acetonitrile, mix Then add 0.1mL trifluoroacetic acid to it and mix well to obtain buffer B); the elution gradient is: within 30 minutes, the proportion of eluent A is gradu...

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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 specifically relates to an antibacterial peptide with antibacterial activity isolated from the spleen of Holstein cows and 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 large-scale use or abuse of antibiotics, the emergence of multi-drug resistant bacteria has brought huge challenges to the treatment of human and animal diseases. At the same time, the problem of antibiotic residues and the side effects of antibiotics pose a huge threat to human and animal health. Therefore, looking for alternatives to antibiotics has become an inevitable choice. [0003] Antimicrobial peptides (AMPs) are an important part of the innate immune system of organisms. The molecular weight of antimicrobial peptides is small, generally composed of 10-60 amino acid residues. In ad...

Claims

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Application Information

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Patent Type & Authority Applications(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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