Feeding antibacterial dodecapeptide from pig lysozyme and preparation method of feeding antibacterial dodecapeptide

An antibacterial peptide and lysozyme technology, applied in the field of bioengineering, can solve the problems of limited application and no bacteriostatic effect, and achieve the effects of simple preparation process and high product purity

Active Publication Date: 2016-09-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, similar to other C-type lysozymes, the antibacterial spectrum of porcine lysozyme is mainly concentrated on Gram-positive bacteria, and has basically no antibacterial effect on Gram-negative bacteria, which also limits its application.

Method used

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  • Feeding antibacterial dodecapeptide from pig lysozyme and preparation method of feeding antibacterial dodecapeptide
  • Feeding antibacterial dodecapeptide from pig lysozyme and preparation method of feeding antibacterial dodecapeptide
  • Feeding antibacterial dodecapeptide from pig lysozyme and preparation method of feeding antibacterial dodecapeptide

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Embodiment 1

[0028] Embodiment 1: the preparation of antibacterial dodecapeptide

[0029] 1. The lysozyme product is fermented by recombinant Escherichia coli BL21 (DE3), and the coding gene (NCBI-ID: 1174173) is digested by BamHI and HindIII and connected with the vector pET-28a(+), transformed into Escherichia coli BL21 (DE3) induced expression. Under the conditions of 25° C. and 200 r / min, 0.1 mmol / L IPTG was used to induce for 8 hours, and the fermentation broth was centrifuged to obtain bacterial cells. Ultrasonic crushing was performed to obtain the recombinant protein inclusion body, and then the inclusion body was renatured, and finally the biologically active porcine lysozyme was obtained after freeze-drying. The fermented product is hydrolyzed by trypsin (15-30mg / g, trypsin / fermented product)) at 35-38°C for 12-16 hours, after the enzymolysis is completed, boil for 10 minutes to kill the enzyme, and the enzymolyzed solution is ultra-filtered (molecular weight cut-off 3000Da) Co...

Embodiment 2

[0037] Embodiment 2: the antibacterial performance detection of antibacterial dodecapeptide

[0038] The antibacterial activity of antibacterial dodecapeptide was determined according to the method in the literature. After secondary activation, the test bacteria were inoculated into a Erlenmeyer flask containing 30mL TSB medium with 1% inoculum and cultivated to OD 600 0.6, take 0.2mL bacterial liquid and mix with 0.4mL TSB medium, add 0.2mL PBS (0.05mol / L, pH 7.0) buffer solution containing antibacterial dodecapeptide (porcine lysozyme as a control) and mix evenly to make the antimicrobial peptide (or control) at a final concentration of 2.5×10- 7 mol / L. After the mixed system was cultured at 37°C and 200r / min for 2 hours, it was diluted and spread on a TSB plate, and counted after the colonies grew. Calculation of the inhibition coefficient log N 0 / N 1 , where N 0 Refers to the number of colonies in the blank group, that is, only PBS solution is added; N 1 is the num...

Embodiment 3

[0042] Embodiment 3: the application of antibacterial dodecapeptide

[0043] The antimicrobial peptide was fused with the porcine lysozyme coding gene to obtain a product with obvious bactericidal effect on both Gram-negative bacteria and positive bacteria, which better made up for the defect of the narrow antibacterial spectrum of porcine lysozyme. The basis for further application is laid.

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Abstract

The invention discloses a feeding antibacterial dodecapeptide from pig lysozyme and a preparation method of the feeding antibacterial dodecapeptide, belonging to the field of bioengineering. The preparation method comprises the steps of preparation of a crude extracting solution, separation of a gel filtration chromatography column Superdux Peptide 10/300GL and separation and purification of a reversed-phase column Phenomenx luna C18. Through LC-MS (Liquid Chromatography-Mass Spectrometry) identification, the amino acid sequence of the antibacterial dodecapeptide is G-V-S-L-A-N-W-V-C-L-A-K; the antibacterial peptide belongs to an alkaline peptide, has remarkable resistance for gram-negative bacteria and is good complementation and expansion for an antibacterial spectrum of the pig lysozyme; through comparison with an antibacterial peptide database and the like, the antibacterial peptide has not been reported. A preparation process disclosed by the invention has the characteristics of simplicity and high efficiency; the purity of a product can reach 95 percent or higher through mass spectrum identification.

Description

technical field [0001] The invention relates to feed antibacterial dodecapeptide derived from porcine lysozyme and a preparation method thereof, belonging to the field of bioengineering. Background technique [0002] For decades, antibiotics have played a positive role in preventing and treating animal diseases, promoting animal growth, and increasing the output of livestock and poultry products by inhibiting the proliferation of pathogenic microorganisms and reducing the toxic effects of their metabolic toxins on animals. However, the long-term use of antibiotics, especially the use of anti-disease drugs and growth-promoting additives in the breeding process, will cause excessive drug residues, environmental pollution in the production area, and lead to a decline in the quality of muscle and processed products. In addition, the long-term use of antibiotics as feed additives in poultry and livestock can cause some bacterial flora to become drug-resistant strains, thus making...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36A23K20/189A23K20/195A61K38/47A61P31/04
Inventor 陆健蔡国林朱德伟
Owner JIANGNAN UNIV
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