Probiotic antimicrobial peptide Enterocin P, and preparation method and application thereof

An antibacterial peptide and probiotic technology, applied in the field of genetic engineering, can solve the problems that restrict the practical application of the antibacterial peptide EnterocinP, the content of the antibacterial peptide is low, and the yield of the antibacterial peptide is low, and achieves significant anti-pathogenic effect, good effect and probiotic effect, and no antibiotics. The effect of drug resistance

Inactive Publication Date: 2016-10-12
源耀生物科技(盐城)股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The content of the antimicrobial peptide Enterocin P in its producing bacteria Enterococcus faecium P13 is extremely low, and the extraction of antimicrobial peptides from the pr

Method used

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  • Probiotic antimicrobial peptide Enterocin P, and preparation method and application thereof
  • Probiotic antimicrobial peptide Enterocin P, and preparation method and application thereof
  • Probiotic antimicrobial peptide Enterocin P, and preparation method and application thereof

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preparation example Construction

[0047] The preparation method of the probiotic antimicrobial peptide Enterocin P of the present invention comprises the following steps:

[0048] (1) Enterocin P gene artificially synthesized with pstI and HindIII restriction sites on both sides has the nucleotide sequence shown in SEQ ID No.3. A gene whose nucleotide sequence is artificially synthesized as shown in SEQ ID NO: 3;

[0049] (2) Construct the pNZ8148-Enterocin P recombinant plasmid capable of highly expressing the antimicrobial peptide Enterocin P; connect the nucleotide sequence shown in SEQ ID No.3 with the linearized pstI and HindIII double-enzyme restriction endonucleases The pNZ8148 plasmid was connected to obtain the pNZ8148-Enterocin P connection product, and the connection product was introduced into Escherichia coli MC1061 to obtain a recombinant plasmid containing pNZ8148-Enterocin P.

[0050] (3) using the pNZ8148-Enterocin P recombinant plasmid to transform the host bacterium to construct a genetical...

Embodiment 1

[0057] Main material used in the present invention

[0058] Strains and vectors: Escherichia coli MC1061, Lactococcus lactis NZ9000 and pNZ8148 plasmid bacteria were purchased from Mo Bi Tec, Germany; DNA Marker was purchased from Shanghai Jierui Co., Ltd.; Listeria, Staphylococcus aureus and Salmonella; probiotic antibacterial peptide Enterocin The P gene was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and transformed into Escherichia coli DH5α; the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0059] Main reagents and kits: M17 broth medium was purchased from Qingdao Haibo Company; restriction enzymes PstⅠ and HindⅢ.T4DNA Ligase.EX Taq were purchased from TaKaRa Biotech Company; chloramphenicol plasmid extraction and DNA gel recovery kit Purchased from Tiangen Company; nisin purchased from Sigma Company.

[0060] Preparation of main reagents:

[0061] GM17: M17 broth medium was autoclaved at 121° C. for 15 minutes, and after cooling...

Embodiment 2

[0074] Preparation of Escherichia coli MC1061 Competent Cells

[0075] 1. 1% LB liquid medium inoculated with MC1061, shake overnight at 37°C;

[0076] 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;

[0077] 3. Centrifuge at 4000g for 10min at 4°C to collect the logarithmic phase cells, discard the supernatant, and collect the bacterial cell pellet;

[0078] 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;

[0079] 5. Centrifuge at 4000g for 5 minutes at 4°C, discard the supernatant, and collect the bacterial cell pellet;

[0080] 6. Use 1 / 100 of the volume of the previous liquid medium containing 0.1M CaCl 2 Solution resuspended bacteria;

[0081] 7. Pipette 200 μL into centrifuge tubes and store at -80°C.

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Abstract

The invention discloses a probiotic antimicrobial peptide Enterocin P, and a preparation method and application thereof. The probiotic antimicrobial peptide Enterocin P gene has nucleotide sequence disclosed as SEQ ID No.1. The mature antimicrobial peptide Enterocin P encoded by the probiotic antimicrobial peptide Enterocin P gene has amino acid sequence disclosed as SEQ ID No.2. The preparation method of the antimicrobial peptide Enterocin P comprises the following steps: (1) synthesizing gene of which the nucleotide sequence is disclosed as SEQ ID NO:3; (2) establishing a recombinant plasmid capable of expressing the antimicrobial peptide; (3) transforming the host bacterium by using the recombinant plasmid to establish a gene engineering bacterium; (4) expressing the antimicrobial peptide by using the gene engineering bacterium; and (5) detecting the antibacterial activity of the antimicrobial peptide. The probiotic antimicrobial peptide Enterocin P has antibacterial activity on pathogenic bacteria, and can be used as an animal feed additive.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a probiotic 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, which seriously threaten 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] An...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/74C07K14/315A23K20/147A23K10/18
CPCC07K14/315C12N15/746C12N2800/101
Inventor 沈城
Owner 源耀生物科技(盐城)股份有限公司
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