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A fusion type antimicrobial peptide and its preparation method and application

An antimicrobial peptide and fusion technology, which is applied in the field of fusion antimicrobial peptide and its preparation and application, can solve the problems of high protease secretion, low gene expression level, etc., to improve immune indicators, not produce drug resistance, improve The effect of weight gain

Active Publication Date: 2015-12-23
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by the high protease secretion and relatively low gene expression level of the original subtilis strain, it has not been widely valued and used by researchers.

Method used

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  • A fusion type antimicrobial peptide and its preparation method and application
  • A fusion type antimicrobial peptide and its preparation method and application
  • A fusion type antimicrobial peptide and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The preparation method of embodiment 1 fusion type antimicrobial peptide GA

[0045] 1. Preparation of SacR regulatory sequence

[0046] (1) Bacillus subtilis genome extraction

[0047] Dip a small amount of frozen Bacillus subtilis WB600 with an inoculation loop, streak on 20 mL of solid LB medium, and culture overnight at 37°C. The next day, a single colony was picked and inoculated in 10 mL of liquid LB medium, shaken at 37°C and 220 rpm for 2 hours, and 5 mL of seed liquid was reserved in aliquots, and all the rest were added to 95 mL of liquid LB medium, shaken at 37°C and 180 rpm for 4 hours until OD600nm≈1.2. The genome of Bacillus subtilis WB600 was extracted using the Axygen Bacterial Genomic DNA Miniprep Kit according to the instructions.

[0048] (2) Using WB600 genome as template PCR to obtain SacB promoter and signal peptide sequence

[0049] According to the SacB sequence published by NCBI:

[0050] 1GATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATG...

Embodiment 2

[0100] The antibacterial property of embodiment 2 fusion type antimicrobial peptide GA

[0101] Take the ATCC25923 (Staphylococcus aureus) and ATCC25922 (Escherichia coli) frozen at -20°C and streak them on the LB plate with the three-line method, culture overnight at 37°C, pick a single colony the next day, and inoculate it in 5 mL of LB liquid culture base test tube, 180rpm, 37°C overnight shaking culture. Plate counting, dilute the bacterial solution to a final concentration of 5 × 105 CFU / mL.

[0102] Select the supernatant fusion antimicrobial peptide GA prepared in Example 1 and obtain the induced expression for 32 hours. After filtering through a 0.22 μm filter membrane, the concentration is recorded as 2C, and partly diluted to a 1 / 2 concentration gradient. 500uL of different concentrations of the supernatant Mix well with 500uL diluted bacterial solution with a concentration of 105CFU / mL, shake culture overnight at 180rpm at 37°C, and the drug concentration of each g...

Embodiment 3

[0109] The application of embodiment 3 fusion type antimicrobial peptide GA as poultry feed additive

[0110] (1) Chick feeding and management

[0111] Sixty healthy 1-day-old male chicks with similar body weight were selected and randomly divided into three groups, 20 in each group. Group I is the experimental group, which is fed with basal diet and added WB600SGA bacteria liquid induced by 2% sucrose after 3 hours of amplification. Group II is the positive control group, which is fed with basal diet and cultured with liquid LB medium 35 hours of Bacillus subtilis WB600 (the final concentration of 2% sucrose was added after expanding the culture for 3 hours), group III was the blank control group, fed the basic diet and added liquid LB liquid medium containing 2% sucrose, and the test period was 21 days .

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Abstract

The invention relates to fusion antibacterial peptide, as well as a preparation method and application thereof. Excretive fusion antibacterial peptide GA is obtained through a plasmid pHYSGA carried by bacillus subtilis WB600. The fusion antibacterial peptide has certain bacteriostatic activity to gram-positive bacterium and gram-negative bacterium and is added to feed as an additive. After eaten by chicks, the fusion antibacterial peptide can increase the growth rate of body mass of the chicks and can improve the immunity indexes of the chicks, and can be an excellent succedaneum of antibiotics in livestock and aquatic feed.

Description

technical field [0001] The present invention relates to a fusion type antibacterial peptide GA, which is composed of GST tagged protein connected to antibacterial peptide AWRK6 through an enterokinase cleavage site, and has certain antibacterial activity on both Gram-positive bacteria and Gram-negative bacteria. Adding the additive to the feed and feeding the chicks proves that the fusion peptide can improve the immune index of the chicks, and is an excellent substitute for antibiotics in poultry, livestock and aquatic feeds. Background technique [0002] At present, in order to improve the immunity of poultry, livestock and aquatic products, most of the professional households in poultry and aquaculture add antibiotics to the feed. The addition of antibiotics not only produces drug resistance, but also has toxic side effects and pollutes the environment. [0003] Antibacterial peptides (ABPs), also known as host defense peptides (HDPs) or antimicrobial peptides (AMPs), are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/75C12P21/00A23K1/16C12R1/125
Inventor 王秋雨金莉莉王宇王铮
Owner LIAONING UNIVERSITY