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Application of recombinant Mytichitin-CB antimicrobial peptide

A technology of antimicrobial peptide and fresh-keeping solution, which is applied in the field of recombinant Mytichitin-CB antimicrobial peptide and its high-efficiency preparation, which can solve the problems of no public use and achieve good temperature stability and good inhibitory effect

Inactive Publication Date: 2019-08-23
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After retrieval, the present invention has no public reports at home and abroad, and no public use at home and abroad

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  • Application of recombinant Mytichitin-CB antimicrobial peptide
  • Application of recombinant Mytichitin-CB antimicrobial peptide
  • Application of recombinant Mytichitin-CB antimicrobial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The preparation of embodiment 1 rMytichitin-CB antimicrobial peptide

[0077] (1) Construction of recombinant expression vector

[0078]Add EcoRI and BamHI restriction sites, start codon, 6×His, and 15 bases of "GATGACGATGACAAG" in sequence at the 5' end of the Mytichitin-CB target gene, and add stop codons, XhoI, KpnI at the 3' end in sequence Restriction sites. Send the gene sequence shown in SEQ ID NO:4 to the company (Suzhou Jinweizhi Biotechnology Co., Ltd.) for synthesis, and connect it to the expression vector pPICZαA through EcoRI and KpnI restriction sites. The connection system is: pPICZαA 20ng, rMytichitin-CB 8ng, T4 ligase 0.8μL, total system 12μL, 4°C, 4h.

[0079] The successfully connected recombinant plasmids were transformed into Escherichia coli competent cells for amplification. Double-digestion verification was performed using restriction endonucleases EcoRI and KpnI.

[0080] Verify the correct strain for sequencing analysis, and the recombinant...

Embodiment 2

[0089] Example 2 rMytichitin-CB antimicrobial peptide performance measurement

[0090] Determination of the minimum inhibitory concentration MIC value - the above-mentioned purified antimicrobial peptide rMytichitin-CB was vacuum freeze-dried into powder, dissolved in sterile Tris-HCl buffer solution (50mM Tris, 100mM NaCl pH 7.4), and the concentrations were respectively Dilute to 2 μg / mL, 4 μg / mL, 6 μg / mL, 12 μg / mL, take 20 μL respectively and add to different wells of a 96-well plate, and make three parallels; set 20 μL of gentamicin at a concentration of 6 μg / mL. The tested bacteria were cultured on a shaker at 37°C to OD 600 1.0-1.5, diluted to 2-5×10 7 CFU / mL, take 100 μL and add to the above wells. Set 100 μL sterile medium plus 20 μL sterile Tris-HCl buffer solution as positive control, 100 μL bacterial solution plus 20 μL sterile Tris-HCl buffer solution as negative control, and do three parallels. After all the addition, seal the 96-well plate with a sealing film ...

Embodiment 3

[0108] Embodiment 3 preservation experiment

[0109] The measurement method of each index:

[0110] (1) Sensory evaluation—according to the provisions on sensory requirements in GB 2707-2016 "National Food Safety Standard for Fresh (Frozen) Livestock and Poultry Products", the samples were placed in a white chassis, and the sensory evaluators followed the table below under natural light Score the sample out of 10 points.

[0111] Sensory score sheet

[0112]

[0113]

[0114] (2) The total number of bacterial colonies—according to the method in GB 4789.2-2016 "Determination of the total number of bacterial colonies in food microbiological examination", with appropriate adjustments, the total number of bacterial colonies is determined for meat samples under different storage times. Randomly sample at regular intervals every day, take 5g meat samples from each treatment group and place them in 50mL centrifuge tubes, homogenize them with 25mL normal saline, dilute the hom...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to application of recombinant Mytichitin-CB antimicrobial peptide (rMytichitin-CB). The amino acid sequence of the antimicrobial peptide is as shown in the sequence table SEQ ID NO:1, and has good temperature stability, pH stability and relative protease stability; the hemolytic activity of human erythrocyte, mouse erythrocyte and chicken erythrocyte is small, the possibility of application in the fields of food, animal husbandry, medicine and the like is achieved; the antimicrobial peptide can be widely applied to preservation and fresh keeping of fresh food, reproduction of microorganisms is inhibited, and the loss of nutrients is lowered.

Description

[0001] Technical field: [0002] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Mytichitin-CB antibacterial peptide (rMytichitin-CB) and a high-efficiency preparation method thereof. [0003] Background technique: [0004] Because antimicrobial peptides (AMPs) have broad-spectrum antibacterial effects, they have inhibitory effects on Gram-positive bacteria, Gram-negative bacteria, and fungi, and can also be antiprotozoal, antiviral, and kill tumor cells in animals without destroying them. Normal cells in animals, and antimicrobial peptides are not easy to produce drug resistance while quickly sterilizing bacteria, will not cause sepsis, and neutralize endotoxins at the same time. They are widely used in medicine, poultry breeding, aquaculture and food field. Mytichitin-CB belongs to the family of cysteine-rich antimicrobial peptides and is one of the defensins among antimicrobial peptides. It was isolated and identified from the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/22C12N15/12C12N15/81A23B4/20C12R1/84
CPCC07K14/43504C12N15/815A23B4/20C12N2800/22A23V2002/00A23V2200/10A23V2250/55
Inventor 孟德梅樊振川石林玥李文娟刘泽松
Owner TIANJIN UNIV OF SCI & TECH
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