Preparation method and application of probiotic antibacterial peptide Enterocin B

A technology of pnz8148-enterocinb and antimicrobial peptides, applied in the field of genetic engineering, can solve the problems of inability to realize large-scale production, low content of antimicrobial peptides, and low production of antimicrobial peptides, and achieve significant anti-pathogenic effects, good effects and probiotic effects, without The effect of antibiotic resistance

Pending Publication Date: 2016-11-09
源耀生物科技(盐城)股份有限公司
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Problems solved by technology

[0006] The content of the antimicrobial peptide Enterocin B in its producing bacterium Enterococcus faecium T136 is extremely low, and the extraction of antimicrobial peptides from the producing bacteria is low in yield, time-consuming, complicated and expensive, and large-scale production cannot be realized, thus restricting the practical application of the antimicrobial peptide Enterocin B
[0007] At present, there is a lack of preparation method and application of a probiotic antibacterial peptide Enterocin B with significant anti-pathogenic effect

Method used

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  • Preparation method and application of probiotic antibacterial peptide Enterocin B
  • Preparation method and application of probiotic antibacterial peptide Enterocin B
  • Preparation method and application of probiotic antibacterial peptide Enterocin B

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preparation example Construction

[0041] The preparation method of the probiotic antimicrobial peptide Enterocin B of the present invention comprises the following steps:

[0042] (1) A gene whose nucleotide sequence is artificially synthesized as shown in SEQ ID NO:3; an artificially synthesized Enterocin B gene with pstI and HindIII restriction sites on both sides has the nucleotide sequence shown in SEQ ID No.3.

[0043] (2) Construct the pNZ8148-Enterocin B recombinant plasmid capable of efficiently expressing the antimicrobial peptide Enterocin B; linearize the pNZ8148 plasmid after the nucleotide sequence shown in SEQID No.3 and the restriction endonucleases pstI and HindIII double enzymes The pNZ8148-Enterocin B ligation product was obtained by ligation, and the ligation product was introduced into Escherichia coli MC1061 to obtain a recombinant plasmid containing pNZ8148-Enterocin B.

[0044] (3) Use the pNZ8148-Enterocin B recombinant plasmid to transform the host bacterium to construct a genetically ...

Embodiment 1

[0050] Main material used in the present invention

[0051] Strains and vectors: Lactococcus lactis NZ9000 and pNZ8148 plasmids were purchased from Mo Bi Tec, Germany; DNAMarker was purchased from Shanghai Jierui Co., Ltd.; Listeria monocytogenes, Staphylococcus aureus and Salmonella were isolated and preserved in our laboratory . Antimicrobial peptide Enterocin B gene was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and transformed into E. coli DH5α; primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0052] Main reagents and kits: M17 broth medium was purchased from Qingdao Haibo Company; restriction enzymes PstⅠ and HindⅢ, T4 DNA ligase, EX Taq were purchased from TaKaRa Biotech Company; chloramphenicol, plasmid extraction and DNA gel The recovery kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; nisin was purchased from Sigma.

[0053] Preparation of main reagents:

[0054] GM17: M17 broth medium was autoclaved ...

Embodiment 2

[0067] Preparation of Escherichia coli MC1061 Competent Cells

[0068] This embodiment prepares Escherichia coli MC1061 competent cells, and the specific steps are as follows:

[0069] 1. 1% LB liquid medium inoculated with MC1061, shake overnight at 37°C;

[0070] 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;

[0071] 3. Centrifuge at 4000g for 10min at 4°C to collect the logarithmic phase cells, discard the supernatant, and collect the bacterial cell pellet;

[0072] 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;

[0073] 5. Centrifuge at 4000g for 5 minutes at 4°C, discard the supernatant, and collect the bacterial cell pellet;

[0074] 6. Use 1 / 100 of the volume of the previous liquid medium containing 0.1M CaCl 2 Solu...

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Abstract

The invention discloses a preparation method and application of a probiotic antibacterial peptide Enterocin B genetically engineered bacterium. A probiotic antibacterial peptide Enterocin B gene has a nucleotide sequence shown as SEQ ID No.1. Mature antibacterial peptide Enterocin B coded by the probiotic antibacterial peptide Enterocin B 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 nuceotide sequence shown as SEQ ID NO:3; (2), establishing recombinant plasmid supportive of efficient expression; (3), establishing a genetically engineered bacterium; (4), utilizing the genetically engineered bacterium to express the antibacterial peptide Enterocin B. The antibacterial peptide enterocin B has high bacteriostatic activity and is safet, nontoxic and free of side effect and antibiotic resistance.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and application of a probiotic antibacterial peptide Enterocin B. 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/74C07K14/315A23K20/195
CPCC07K14/315C12N15/74C12N2800/101
Inventor 杨旭方华季春源沈城郭子好
Owner 源耀生物科技(盐城)股份有限公司
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