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Method and application for preparing bacteria simulacrum by using antibacterial peptide

A technology of antimicrobial peptides and bacteria, applied in the field of genetic engineering, can solve the problems of inability to prepare, complicated operation, time-consuming and labor-intensive cracking carrier, etc., and achieve good immune effect and simple operation

Inactive Publication Date: 2010-03-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the traditional methods for the preparation of bacterial ghosts have the following disadvantages: To prepare the bacterial ghosts of different bacteria, different lysis carriers need to be constructed, and if there is no corresponding carrier, the bacterial ghosts of the bacteria cannot be prepared; the construction of the lysis carrier is time-consuming and laborious, and the operation is complicated; Only limited to the preparation of gram-negative bacterial shadows, limiting the scope of use
[0007] At present, there is no report on the use of antimicrobial peptide technology in the preparation of bacterial shadows at home and abroad

Method used

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  • Method and application for preparing bacteria simulacrum by using antibacterial peptide
  • Method and application for preparing bacteria simulacrum by using antibacterial peptide
  • Method and application for preparing bacteria simulacrum by using antibacterial peptide

Examples

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Effect test

Embodiment 1

[0026] Preparation of Actinobacillus pleuropneumoniae, Bacillus subtilis and Escherichia coli O by antibacterial peptide Tachyplesin I 138 Shroom

[0027] The antimicrobial peptide selected in this embodiment is Tachyplesin I; the bacteria selected are Actinobacillus pleuropneumoniae (Gram-negative bacteria), Bacillus subtilis (Gram-positive bacteria) and Escherichia coli O 138 (Gram-negative bacteria), the above bacteria were all preserved in our laboratory.

[0028] The DNA sequence of Tachyplesin I is:

[0029] aaatggtgct tccgtgtttg ctaccgtggt atctgctacc gtcgttgccg t 51

[0030] The amino acid sequence of Tachyplesin I is:

[0031] Lys Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys Arg

[0032] 1. Synthesis and expression of antimicrobial peptides

[0033] The complete sequence of the antimicrobial peptide Tachyplesin I was synthesized with an automatic peptide synthesizer APEX396, the N-terminal was free, and the C-terminal was α-amidated. 18 Desalting a...

Embodiment 2

[0039] Preparation of Actinobacillus pleuropneumoniae and Escherichia coli O by Antibacterial Peptide Catesbeianin-1 138 Shrooms

[0040] The antibacterial peptide that present embodiment selects is Catesbeianin-1; 138 (Gram-negative bacteria), the above bacteria were all preserved in our laboratory.

[0041] The DNA sequence of Catesbeianin-1 is:

[0042] atgatgaggg tgatgaggag gaagactaaa gttatttggg aaaaaaagga ctttatgga 60

[0043]ttatacagta ttgattga 78

[0044] The amino acid sequence of Catesbeianin-1 is:

[0045] Met Met Arg Val Met Arg Arg Lys Thr Lys Val Ile Trp Glu Lys Lys

[0046] Asp Phe Ile Gly Leu Tyr Ser Ile Asp

[0047] 1. Synthesis and expression of antimicrobial peptides

[0048] The complete sequence of the antimicrobial peptide Catesbeianin-1 was synthesized with an automatic peptide synthesizer APEX396. The N-terminal was free and the C-terminal was α-amidated. 18 Desalting and purification by column chromatography; eukaryotic expression of antimicrobi...

Embodiment 3

[0054] Preparation of Actinobacillus Pleuropneumoniae Ghosts Using Antimicrobial Peptide MAP

[0055] The antimicrobial peptide used in this example is MAP; the bacteria used is Actinobacillus pleuropneumoniae (preserved in our laboratory).

[0056] The amino acid sequence of MAP is:

[0057] Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys Leu Ala 18

[0058] 1. Synthesis of Antimicrobial Peptides

[0059] The complete sequence of the antimicrobial peptide MAP was synthesized with the automatic peptide synthesizer APEX396, the N-terminus is free, and the C-terminus is α-amidation modification. 18 Desalted and purified by column chromatography.

[0060] 2. Preparation of Bacterial Ghosts

[0061] After enriching the bacterial strain overnight, inoculate it in the culture medium at 0.5%, shake culture at 37°C when the OD600 is 0.4-0.6, add the synthetic antimicrobial peptide MAP to a final concentration of 41.7ug / mL, and act overnight at 4°C or Treat at room...

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Abstract

The invention discloses a method and an application for preparing bacteria simulacrum by using antibacterial peptide, comprising the following steps of: when culturing the bacteria strains to a logarithm growth period (OD(600) is 0.4-0.6), adding the antibacterial peptide, reacting for one night at the temperature of 4 DEG C or reacting for 2-4 hours at the room temperature; after reaction, centrifugally collecting thalli and repeatedly washing the thalli twice by physiological saline, thus preparing the bacteria simulacrum. The antibacterial peptide is used for replacing the traditional method to prepare the bacteria simulacrum; the new method for preparing the bacteria simulacrum obtains good effect, has simple operation, can be widely used for the preparation of bacteria simulacrum, andthe prepared bacteria simulacrum has good immunity effect and can be used as a new candidate vaccine.

Description

technical field [0001] The invention relates to a method and application for preparing bacterial ghosts by using antibacterial peptides, belonging to the field of genetic engineering. Background technique [0002] Antibacterial peptides (antibacterial peptides), in a broad sense, refers to a class of small molecule peptides that exist in organisms to resist external microbial invasion and eliminate mutant cells in the body. Defensive polypeptides are an important part of biological innate immune defense system, which are called "second defense system" and "second immune system". Under the induced conditions, the animal immune defense system produces a kind of defensive cationic peptide active substances against exogenous pathogens, which is an important part of the biological innate immune defense system. [0003] Antimicrobial peptides have relatively small molecular weight, generally less than 10kDa, and are encoded by genes and synthesized by ribosomes. Distortion effec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61K39/108A61K47/46A61P31/04
CPCY02A50/30
Inventor 雷连成杜崇涛韩文瑜冯新谢芳江丽娜
Owner JILIN UNIV