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Preparation method and application of pig small intestine compound antibacterial peptide

A composite antibacterial and pig small intestine technology, applied in the fields of biological products, animal husbandry and veterinary medicine, and biopharmaceuticals, can solve the problems of low concentration of antibacterial peptide PR39 in pig small intestine, plasmolysis, shrinkage and death, and unsuitability for large-scale production. Immune stress, high yield, high concentration effect

Active Publication Date: 2015-10-28
派生特(福州)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Microorganisms in a hypertonic solution are prone to plasmolysis, shrinkage and death; on the contrary, if they are in a hypotonic solution, they are prone to absorb water, swell and die
[0011] The patent application number 201210305972.2 discloses a preparation method of porcine small intestine antibacterial peptide PR39, which uses ultrasonic cracking to release proteins in the intestinal mucosa, and uses separation column chromatography to remove most of the foreign proteins to obtain antibacterial peptides. A single PR39 antimicrobial peptide was extracted from this method, the process is complicated, the yield is not high, and it is not suitable for large-scale production, and the concentration of the obtained porcine small intestine antimicrobial peptide PR39 is not high

Method used

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  • Preparation method and application of pig small intestine compound antibacterial peptide
  • Preparation method and application of pig small intestine compound antibacterial peptide
  • Preparation method and application of pig small intestine compound antibacterial peptide

Examples

Experimental program
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Effect test

Embodiment 1

[0038] 1. Preparation of complex antimicrobial peptides from porcine small intestine

[0039](1) Rinse 7.5Kg of fresh pig small intestine (including duodenum and jejunum) with deionized water, remove the content, serosa and fat, rinse thoroughly with water for injection, freeze, shred, and mince The pig small intestine colloid was obtained by machine twisting twice;

[0040] (2) Homogenize the porcine small intestine colloid through a colloid mill to obtain 3.8 L of tissue homogenate, then dispense the homogenate into non-pyrogenic bottles, freeze at -40°C, thaw with running water at 37°C, and freeze and thaw three times repeatedly. The homogenate was boiled in water at 100°C for 15 minutes, then cooled rapidly to 4°C;

[0041] (3) At 4°C, 3.8L of the freeze-thawed homogenate and 3.8L of acetic acid solution with a concentration of 5.00% were stirred and leached for 12 hours, and the mixture after stirring and leaching was centrifuged at 8000rpm at 4°C for the first time. Ce...

Embodiment 2

[0101] (1) Rinse 150Kg of fresh pig small intestine (including duodenum, jejunum, and ileum) with deionized water, remove the content, serosa and fat, rinse thoroughly with water for injection, freeze, cut into pieces, and twist The pig small intestine colloid was obtained by first twisting the meat machine for 3 times;

[0102] (2) Homogenize the porcine small intestine colloid through a colloid mill to obtain 80 L of tissue homogenate, then distribute the homogenate into non-pyrogenic bottles, freeze at -40°C, thaw with running water at 37°C, and freeze and thaw five times repeatedly. The slurry was boiled in water at 100°C for 20 minutes, then cooled rapidly to 4°C;

[0103] (3) Under the condition of 4°C, 80L of the freeze-thawed homogenate and 80L of acetic acid solution with a concentration of 5.00% were stirred and leached for 10 hours, and the mixture after stirring and leaching was centrifuged for the first time at 4°C at 8000rpm for 25min. , 93L of supernatant was o...

Embodiment 3

[0120] (1) Rinse 7.5Kg of fresh pig small intestine (including duodenum and ileum) with deionized water, remove the content, serosa and fat, rinse thoroughly with water for injection, freeze, cut into pieces, and grind the meat The pig small intestine colloid was obtained by machine twisting once;

[0121] (2) Homogenize the porcine small intestine colloid through a colloid mill to obtain 3.8 L of tissue homogenate, then distribute the homogenate into non-pyrogenic bottles, freeze at -40°C, thaw with running water at 37°C, and freeze and thaw 6 times repeatedly. The homogenate was boiled in water at 100°C for 25 minutes, then rapidly cooled to 4°C;

[0122] (3) Stir and leach 3.8L of the freeze-thawed homogenate and 3.80L of acetic acid solution with a concentration of 5.00% at 1°C for 8 hours, and centrifuge the mixture after stirring and leaching at 7000rpm at 4°C for the first time. Centrifuge for 20 minutes to obtain 4.4L of the first supernatant, store at -20°C, and prep...

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Abstract

The invention discloses a preparation method of a recombinant antimicrobial peptide extracted from a small intestine of a pig and an application thereof. The preparation method comprises the following steps: mashing, homogenizing and repeatedly freezing and thawing the small intestine tissue of the pig with a meat grinder and a colloid mill, and preliminarily crushing cells; then performing the water boiling heat treatment, and extracting with acetic acid solution to obtain a large amount of antimicrobial peptides; centrifuging to enable the quantity of other proteins to be reduced greatly, filtering the supernate obtained through centrifuging by a tangential flow micro-filtration membrane and a tangential flow ultrafiltration membrane sequentially, adjusting the pH value, filtering the supernate by a tangential flow nanofiltration membrane, concentrating and dialyzing to desalt, and disinfecting after adjusting the osmotic pressure to obtain the recombinant antimicrobial peptide extracted from the small intestine of the pig. According to the invention, the application of the recombinant antimicrobial peptide adopts effects of the antimicrobial peptide on aspects of mucosal immunity, immunopotentiators, novel antibiotics and the like, and the mucosal immunity character of the antimicrobial peptide can play an important role in the prevention and control of transmissible gastroenteristis, epidemic diarrhea and mycoplasma gallisepticum; the antimicrobial peptide replaces traditional antibiotics, and is remarkable in effect.

Description

technical field [0001] The invention belongs to the fields of animal husbandry and veterinary medicine, biological products, and biopharmaceuticals, and specifically relates to a preparation method and application of a pig small intestine compound antibacterial peptide. Background technique [0002] Antimicrobial peptides are short peptides produced by biological organisms during defense against pathogenic microorganisms and some malignant cells. Some international literatures use "antimicrobial peptides" (AMPs) or "peptide antibiotics". Antimicrobial peptides have antibacterial effects, and some also have antiprotozoal, antifungal, antiviral, and inhibitory or killing effects on tumor cells and parasites. It has the characteristics of small molecular weight (3-10 KD), good water solubility, heat resistance, and no immunogenicity. It can become a new antibacterial, antiviral, and anticancer drug with high efficiency, low toxicity, and no residual hazards. [0003] At prese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/00A61K35/38A61K39/39A61P31/04A61P37/04
Inventor 徐磊刘友霖曾辉荣
Owner 派生特(福州)生物科技有限公司
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