Antibacterial peptide Enterocin P and preparation method and application thereof

A technology of antimicrobial peptides and host bacteria, applied in the field of genetic engineering, to achieve strong antibacterial activity, no antibiotic resistance, good effect and probiotic effect

Inactive Publication Date: 2016-08-17
源耀生物科技(盐城)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is a lack of an antibacterial peptide Enterocin P wi

Method used

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  • Antibacterial peptide Enterocin P and preparation method and application thereof
  • Antibacterial peptide Enterocin P and preparation method and application thereof
  • Antibacterial peptide Enterocin P and preparation method and application thereof

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preparation example Construction

[0049] The preparation method of the antimicrobial peptide Enterocin P of the present invention, comprises the steps:

[0050] (1) The gene whose nucleotide sequence is artificially synthesized as shown in SEQ ID NO: 3; the artificially synthesized Enterocin P gene with restriction sites containing Sac I and Hind III on both sides has the nucleotide sequence shown in SEQ ID No.3 .

[0051] (2) Construct the pMG36e-Enterocin P recombinant plasmid capable of highly expressing the antimicrobial peptide Enterocin P; connect the nucleotide sequence shown in SEQ ID No.3 with restriction endonucleases Sac I and Hind III and linearize after double digestion The pMG36e plasmid was ligated to obtain the pMG36e-Enterocin P ligation product, and the ligation product was introduced into Escherichia coli DH5α to obtain a recombinant plasmid containing pMG36e-Enterocin P.

[0052] (3) using the pMG36e-Enterocin P recombinant plasmid to transform the host bacterium to construct a genetically...

Embodiment 1

[0059] Main material used in the present invention

[0060] Strains and vectors: Escherichia coli MC1061, Lactococcus lactis MG1363 and pMG36e plasmid bacteria were purchased from Mo Bi Tec, Germany; DNA Marker was purchased from Shanghai Jierui Co., Ltd.; Listeria, Staphylococcus aureus and Salmonella were preserved in our laboratory ; The antibacterial peptide Enterocin P gene was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and transferred into Escherichia coli DH5α; primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0061] Main reagents and kits: M17 broth medium was purchased from Qingdao Haibo Company; restriction enzymes Sac Ⅰ and HindⅢ, T4 DNA Ligase, EX Taq were purchased from TaKaRa Biotech Company; erythromycin, plasmid extraction and DNA gel The recovery kit was purchased from Tiangen Company; nisin was purchased from Sigma Company.

[0062] Preparation of main reagents:

[0063] GM17: M17 broth medium was autoclaved at 121° C. ...

Embodiment 2

[0076] Preparation of Escherichia coli DH5α Competent Cells

[0077] 1. 1% LB liquid medium inoculated with DH5α, shake overnight at 37°C;

[0078] 2. Take 1% of the overnight culture solution and inoculate it in LB liquid medium, culture it with shaking at 37°C for 4 hours (the bacteria are in the logarithmic growth phase at this time), and place it on ice for 10 minutes;

[0079] 3. Centrifuge at 4000g for 10min at 4°C to collect the logarithmic phase cells, discard the supernatant, and collect the bacterial cell pellet;

[0080] 4. Use pre-cooled 0.1M CaCl 1 / 10 of the volume of the previous liquid medium 2 Bacteria, placed on ice for 30 minutes;

[0081] 5. Centrifuge at 4000g for 5 minutes at 4°C, discard the supernatant, and collect the bacterial cell pellet;

[0082] 6. Use 1 / 100 of the volume of the previous liquid medium containing 0.1M CaCl 2 Solution resuspended bacteria;

[0083] 7. Pipette 200 μL into centrifuge tubes and store at -80°C.

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Abstract

The invention discloses a preparation method and application of antibacterial peptide Enterocin P. An antibacterial peptide Enterocin P gene has a nucleotide sequence shown as SEQ ID No.1. A mature antibacterial peptide Enterocin P coded by the antibacterial peptide Enterocin P gene has an amino acid sequence shown as SEQ ID No.2. The preparation method includes following steps: (1), artificially synthesizing a gene with a nucleotide sequence shown as SEQ ID NO:3; (2), building recombinant plasmid capable of realizing efficient expression; (3), building a gene engineering bacterium of lactococcus lactis NZ900; (4), utilizing the gene engineering bacterium to express the antibacterial peptide; (5), detecting antibacterial activity of the antibacterial peptide. The antibacterial peptide has high antibacterial activity to pathogenic bacteria and is safe, nontoxic and free of side effect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an antibacterial peptide Enterocin P and a preparation method and application thereof. Background technique [0002] In recent years, the extensive abuse of antibiotics in animal breeding has led to the emergence of antibiotic-resistant strains, drug residues, environmental pollution and ecological damage, and many other problems, seriously threatening human health and the development of animal husbandry. Therefore, it is extremely urgent to find a new antibacterial agent that can replace antibiotics. Antimicrobial peptides have the advantages of good antibacterial effect, good thermal stability, non-toxicity, no residue, no side effects, safety and high efficiency, no pollution to the environment, and less likely to produce bacterial resistance. It is expected to become an effective method to solve the problem of antibiotic abuse. [0003] Antimicrobial ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/74C07K14/315A23K20/195
CPCC07K14/315C12N15/74C12N2800/101
Inventor 沈城
Owner 源耀生物科技(盐城)股份有限公司
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