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Preparation method of antibacterial peptide gene engineering strain

A technology of genetically engineered strains and antimicrobial peptides, applied in the field of preparation of genetically engineered strains of antimicrobial peptides, can solve the problems of high production costs, small quantities, and inability to meet the needs of applications

Inactive Publication Date: 2010-10-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Antimicrobial peptides isolated and purified from insects and other organisms are small in quantity and high in production costs, which cannot meet the needs of applications

Method used

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  • Preparation method of antibacterial peptide gene engineering strain

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Effect test

preparation example Construction

[0019] The preparation method of antimicrobial peptide genetically engineered bacterial strain comprises the following steps:

[0020] 1) According to the antimicrobial peptide Cecropin A gene sequence with the registration number AAA29185, the whole sequence was synthesized, and at the same time, histidine tag sequence and restriction enzyme cutting sites NotI and XhoI were respectively introduced to obtain the antimicrobial peptide Cecropin A gene synthesis product;

[0021] 2) Cloning the antimicrobial peptide Cecropin A gene synthesis product into the pGEM-T vector to obtain the recombinant vector pGEM-T-CEC, and transferring the recombinant vector pGEM-T-CEC into Escherichia coli DH5α to obtain DH5α (pGEM-T-CEC) strains;

[0022] 3) Extract the recombinant vector pGEM-T-CEC plasmid from the DH5α (pGEM-T-CEC) strain, use NotI and XhoI to double-digest the plasmid pGEM-T-CEC to obtain the target fragment Cecropin A; similarly digest the yeast shuttle vector pPIC9k to make ...

Embodiment 1

[0032] 1. Synthesis of antibacterial peptide Cecropin A gene sequence, introducing enzyme cutting sites NotI and XhoI during design:

[0033] ctcgagaagtggaagttgtttaagaaaattgaaaaggttggtcaaaacattagagatggtattattAaggctggtccagctgttgctgtttgttggtcaagctactcaaattgctaaggcggccgc

[0034] (a) Sequence features:

[0035] Length: 125 base pairs

[0036] Type: nucleic acid

[0037] Chain type: double chain

[0038] Topology: Linear

[0039] (b) Molecular type: double-stranded DNA

Embodiment 2

[0041] Ligation of Antimicrobial Peptide Cecropin A Gene Sequence and pGEM-T Intermediate Vector

[0042](1) The synthetic product was cloned into the pGEM-T vector: take 1 μg of the antimicrobial peptide Cecropin A prepared in Example 1 and connect it to the T vector according to the following system (using Takara cloning kit)

[0043] 10×ligation Buffer 1μL

[0044] Cecropin A 5 μL

[0045] pGEM-T vector 1 μL

[0046] T4 DNA ligase 1 μL

[0047] ddH2O Up to 10μL

[0048] (2) Transform the recombinant vector pGEM-T-CEC into Escherichia coli DH5α:

[0049] a) Immediately place the cryopreserved E. coli DH5α competent cells in an ice bath, then add 15 μL of the ligation product to 100 μL of the competent cells, ice bath for 30 minutes, and then quickly place them in a water bath at 42°C for 90 seconds for heat shock , placed in an ice bath for 2 minutes immediately, then added to 800 μL of SOC medium, incubated at 170 rpm at 37°C for 50 minutes, then centrifuged the cultur...

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Abstract

The invention discloses a preparation method of an antibacterial peptide gene engineering strain. The method comprises the following steps of: firstly, designing a new antibacterial peptide gene according to the characteristic of an antibacterial peptide Cecropin A amino acid sequence, adding a histidine marked sequence at the 5'end of the sequence, and respectively adding Xho I and Not I restriction enzyme cutting site sequences at the two ends to synthesize a sequence of the antibacterial peptide gene; secondly, cloning the obtained product to a T vector; thirdly, constructing a target gene into a yeast expression vector pPIC9k to construct a recombination vector pPIC-CEC containing the target gene; and fourthly, transforming the recombination vector pPIC-CEC into pichiapastoris for expressing to form a antibacterial peptide gene transformation pichiapastoris engineering strain GS115 (pPIC-CEC). The strain can express and generate gene engineering antibacterial peptide Cecropin A, has low cost, can ferment in large scale, and is convenient to produce and easy to realize industrialization. The invention has the advantages of simple operation method, easy observation and good repeatability. The expressed product can be applied to biological control of fruits after picking and can bring certain economic benefit.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing an antimicrobial peptide genetically engineered bacterial strain. Background technique [0002] Antimicrobial peptides, as the name suggests, are small molecule polypeptides that can inhibit or kill bacteria, fungi, and other microorganisms, as well as certain insects, animal and plant cells, and their molecular weight is usually below 10KD. They have the characteristics of small molecular weight, good water solubility, strong thermal stability, and broad antibacterial spectrum (Rao et.al, 1995). In addition, antimicrobial peptides have the advantages of small molecular weight, thermal stability, good water solubility, and no side effects on humans, and have broad application prospects in the fields of medicine, agriculture, and food industry. In the food industry, antimicrobial peptides are mainly used as food preservation preservatives. At present, most of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/11C12N15/81A23B7/154C12R1/84
Inventor 郑晓冬任雪艳陈新爱
Owner ZHEJIANG UNIV
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