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Preparation method and application of bombyx mori antibacterial peptide BMGlvA2 recombinant protein

A silkworm antibacterial peptide and recombinant protein technology, applied in the fields of genetic engineering and molecular biology, can solve the problems of restricting industrial production and application, high cost, etc.

Pending Publication Date: 2021-09-24
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production methods studied mainly include separation and extraction. This high-cost production severely limits its industrial production and application.

Method used

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  • Preparation method and application of bombyx mori antibacterial peptide BMGlvA2 recombinant protein
  • Preparation method and application of bombyx mori antibacterial peptide BMGlvA2 recombinant protein
  • Preparation method and application of bombyx mori antibacterial peptide BMGlvA2 recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Construction of antimicrobial peptides BMGlvA2 engineering bacteria

[0069] Determining (1) the amino acid sequence of the antimicrobial peptide BMGlvA2 recombinant protein

[0070] The amino acid sequence silkworm Bombyx mori (domestic silkworm) antimicrobial peptides derived from the amino acid sequence disclosed in GenBank BMGlvA2 No.AB239448 original amino acid sequence, by error prone PCR and screened for the recombinant protein BMGlvA2 antimicrobial peptide, such as SEQ ID NO. 1 shown in FIG.

[0071] (2) Construction of recombinant expression vector

[0072] The recombinant protein synthesis BMGlvA2 antimicrobial peptide sequences using PCR fidelity synthase was amplified by PCR reaction. PCR amplification procedure is 95 ℃ denaturation 5min, 95 ℃ denaturation 30s, 60 deg.] C annealing 30s, extension 30s 72 ℃, 35 cycles, then extended 10min 72 ℃. The amplified fragment Trichoderma expression vector into E. coli DH5α competent cells in vitro recombinant connected, an...

Embodiment 2

[0074] BMGlvA2 antimicrobial peptide expression of recombinant proteins in Trichoderma reesei in

[0075] (1) transforming the recombinant plasmid in Trichoderma reesei

[0076] The T. reesei host strains in solid medium plates PDA + U subcultured conditions were 30 ℃ 5-6 days. The mature T. reesei host strain was inoculated to YEG culture medium, as an expression host 20h at 30 ℃ 180rpm conditions.

[0077] Mature expression host cultured mycelium was collected, and rinsed with sterile water. Weigh the washed mycelia to 1-2g standby 100mL Erlenmeyer flask, which was cleaved under the conditions of 30 ℃ 90rpm, every 30min number of protoplasts prepared samples under a microscope using a hemocytometer calculated until it reaches 10 8 CFU / mL stop cracking. The filtrate was collected after lysis was filtered, centrifuged and washed continuously with sorbitol solution, Trichoderma protoplasts were collected.

[0078] (2) The recombinant expression plasmid flask fermentation of Trich...

Embodiment 3

[0082] Detecting the recombinant protein antibacterial activity of antimicrobial peptides of BMGlvA2

[0083] (1) Determination of antibacterial peptide BMGlvA2 recombinant protein in bacteria MIC

[0084] MIC were measured by micro dilution method. Including bacterial suspension was prepared, antimicrobial peptides processing BMGlvA2 recombinant protein detection.

[0085] Preparation of the bacterial suspension: E. coli, Salmonella grown to log phase growth, the same Clostridium perfringens grown to log phase growth in BHI broth in a LB liquid medium. The three kinds of bacteria cells were respectively diluted with the appropriate liquid medium until the concentration was 10 5 CFU / ml.

[0086] Peptide BMGlvA2 processing the recombinant protein: The fermentation product was sterilized by filtration and lyophilized to a powder. Weighed amount of sample powder was reconstituted in sterile water. In each 96-well plate was added 100μL of the appropriate liquid media pathogens, the ...

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Abstract

The invention provides a preparation method and application of a bombyx mori antibacterial peptide BMGlvA2 recombinant protein, and the amino acid sequence of the bombyx mori antibacterial peptide BMGlvA2 recombinant protein is as shown in SEQ ID NO. 1. The invention relates to the field of gene engineering, and an engineering strain Trichoderma reesei is used as an expression host to realize expression of the bombyx mori antibacterial peptide BMGlvA2 recombinant protein. Escherichia coli, salmonella and clostridium perfringens are used as indicator bacteria at the same time for the first time, and it is identified that the antibacterial peptide fermentation product have obvious antibacterial activity on the indicator bacteria. The invention also characterizes the self properties of temperature tolerance, digestive enzyme tolerance, hemolytic activity and the like of the antibacterial peptide, and also preliminarily provides an inhibition mechanism of the antibacterial peptide on clostridium perfringens. The bombyx mori antibacterial peptide BMGlvA2 provided by the invention is beneficial to further development and application in the field of feeds.

Description

Technical field [0001] The invention belongs to the field of genetic engineering and molecular biology, and in particular, the present invention relates to the preparation method of BMGLVA2 recombinant protein, and its application. Background technique [0002] With the continuous development of the aquaculture, excessive use of antibiotics causes the anti-pharmaceutical strain to make anti-borne ban policies. Because antimicrobial peptides have a wide range of sources and strong antibacterial activity, antibacterial peptides have been attracted to become potential antibiotic substitutes. The antibacterial peptide that has been reported has been shown to be inhibited to Gram-positive and negative bacteria, fungi, etc., which is the most widely used in breeding industry. The most extensive insect antibacterial peptide is mainly from silkworms, which belong to silkworm. The family antibacterial peptide has a unique α-helical structure and sequence, rich in specific amino acids. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/80A23K20/147
CPCC07K14/43586C12N15/80A23K20/147
Inventor 牟海津梁青平刘哲民
Owner OCEAN UNIV OF CHINA
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