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Recombinant antimicrobial peptide (AMP) as well as genetic engineering preparation method and application thereof

A technology of antimicrobial peptides and genes, applied in the field of recombinant antimicrobial peptides, can solve the problems of inability to meet safety and green feed additive production requirements, high cost of chemically synthesized antimicrobial peptides, inconvenient large-scale production and use, etc., to improve the microbial system of the digestive tract, Effects of activating animal immunity and improving production performance

Inactive Publication Date: 2011-10-19
SHENZHEN SUNSMILE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Due to the low efficiency and time-consuming preparation of PMAP-36 from natural sources, the yield of purified antimicrobial peptides is extremely low, time-consuming and laborious, and separation and purification are difficult, resulting in high production costs and inconvenient for large-scale production and use
However, the cost of chemically synthesizing antimicrobial peptides is high, and toxic organic substances are often added during the preparation process, which cannot meet the production requirements of safe and green feed additives

Method used

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  • Recombinant antimicrobial peptide (AMP) as well as genetic engineering preparation method and application thereof
  • Recombinant antimicrobial peptide (AMP) as well as genetic engineering preparation method and application thereof
  • Recombinant antimicrobial peptide (AMP) as well as genetic engineering preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Obtaining and cloning construction of PMAP-36 gene

[0056] 1. Acquisition of PMAP-36 gene

[0057] refer to LS (Invitrogen) kit manual, extracting genomic RNA from sow femur bone marrow. The specific steps are:

[0058] 1) Take sow femur bone marrow, add a little ddH2O to grind. Take a small amount of sample into an EP tube, add 900 μL of TRIzol reagent, mix it evenly with a gun, and let it stand at 15-30°C for 5 minutes.

[0059] 2) Add 200 μL of chloroform to the EP tube, shake vigorously for 15 seconds, mix well, and let stand at 15-30°C for 2-15 minutes.

[0060] 3) Centrifuge at 12000g for 15min at 4°C.

[0061] 4) Take the colorless aqueous phase (upper solution) into a new EP tube, add 0.5ml of pre-cooled isopropanol, mix well, and place at -20°C for more than 10 minutes to fully precipitate the nucleic acid, and discard the lower organic phase.

[0062] 5) Precipitate RNA by centrifugation at 12000 g for 10 min at 4°C.

[0063] 6) Discard ...

Embodiment 2

[0090] Embodiment 2: Construction and expression of recombinant engineering bacteria

[0091] 1. Construction of Saccharomyces cerevisiae expression vector

[0092] The positive recombinant plasmid and the yeast shuttle plasmid pVT102U / α were digested with XbaI and HindIII. The double enzyme digestion reaction system is as follows:

[0093]

[0094]

[0095] Recover the product after double enzyme digestion, mix the antimicrobial peptide gene and the carrier at a molar ratio of 5-10:1, add 1 μl of T4 DNA ligase and 2 μl of 10× ligation buffer, and perform ligation reaction overnight in a water bath at 16°C , to construct the recombinant plasmid pVT-PMAP-36.

[0096] Take 80 μl of competent yeast cells and mix them with 5-10 μl of recombinant plasmid (about 10 μl), transfer them to an ice-precooled 0.2 cm electroporation cell, place the mixture on ice for 5 min; 1500 V, 5 ms electric shock, then add 1 ml to recover The culture solution was cultivated in an incubator at...

Embodiment 3

[0098] 1. Adding recombinant antimicrobial peptide PMAP-36 to sow diet significantly increased sow colostrum IgG level

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Abstract

The invention provides a preparation method and application of a recombinant antimicrobial peptide (AMP) and belongs to the technical field of biological genetic engineering. The method comprises: amplification of an antibacterial peptide gene, gene cloning, transformation of beer yeast and efficient expression and purification of recombinant protein. The recombinant AMP provided by the invention is AMP-36 of the porcine Cathelicidins family, and the protein of the recombinant AMP contains short peptide shown by the sequence ID No.2. By utilizing the method provided by the invention, the prepared recombinant AMP can be used for producing a feed additive, a veterinary medicine and a preservative and preparing Gram-negative bacteria, Gram-positive bacteria and medicaments for diseases caused by fungi or virus.

Description

【Technical field】 [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a recombinant antibacterial peptide, a method for preparing genetically engineered bacteria and its application. 【Background technique】 [0002] At present, antimicrobial / antibacterial peptides (AMP / ABP) has become a hotspot in the research of immunology and molecular biology. Antimicrobial peptides are a general term for a class of cationic short peptides with antibacterial activity. They are an important part of biological innate immunity and widely exist in various bacteria, plants, animals and humans. Antimicrobial peptides have high-efficiency and broad-spectrum antimicrobial activity characteristics, which make them have a strong killing effect on Gram-positive and negative bacteria. In addition, they also have fungicidal, protozoan, antiviral activity, and tumor cell killing. [0003] In recent years, due to the excessive abuse of antibiotics ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/12C12N15/81C07K14/47A23K1/16A23L3/3526A01N63/04A61K38/17A01P1/00A01P3/00A61P31/04A61P31/10A61P31/12A61P33/02A61P35/00C12R1/865A23K20/147
Inventor 彭永鹤李永新林美娟
Owner SHENZHEN SUNSMILE BIOTECH
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