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Preparation method of artificial antibacterial peptide G3

An antimicrobial peptide and artificial technology, which is applied in the fields of genetic engineering and molecular biology, can solve the problems of complex process, high cost, and low content of antimicrobial peptide, and achieve the effect of simple separation and purification process, low production cost and high expression efficiency

Pending Publication Date: 2022-04-05
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of antimicrobial peptides in animals is very small
Extracting antimicrobial peptides from animals has low yield, time-consuming, complex process, and high cost, making it impossible to achieve large-scale production
In addition, antimicrobial peptides usually have a complex and repetitive structure. Although they can also be synthesized by chemical methods, the synthesis efficiency is low and the cost is high.
At present, most of the genetically engineered drugs that have entered clinical application are produced using prokaryotic expression systems, but due to the killing effect of antimicrobial peptides on bacteria, it is not possible to directly express biologically active antibacterial peptides using prokaryotic expression systems, and if fusion proteins are used expression, will bring a lot of trouble to the post-processing of the expression product
Therefore, the large-scale production of antimicrobial peptides has become the biggest obstacle restricting the practical application of antimicrobial peptides.

Method used

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  • Preparation method of artificial antibacterial peptide G3
  • Preparation method of artificial antibacterial peptide G3
  • Preparation method of artificial antibacterial peptide G3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A preparation method of artificial antibacterial peptide G3, the steps are as follows:

[0040] 1. Construction of recombinant expression vector

[0041] According to the amino acid sequence of the artificial antimicrobial peptide G3, the nucleotide sequence encoding the antimicrobial peptide is designed as the coding gene sequence of the antimicrobial peptide. Wherein, the amino acid sequence of the artificial antimicrobial peptide G3 is shown in SEQ ID NO:1; the nucleotide sequence encoding the antibacterial peptide is shown in SEQ ID NO:2.

[0042] GIIKKIIKKIIKKI (SEQ ID NO: 1);

[0043] 5'-GGTATCATCAAAAAAATCATCAAAAAAATCATCAAAAAAATC-3' (SEQ ID NO: 2);

[0044] Utilize the method of fusion expression of glutathione-S-transferase (GST) label and target gene, obtain fusion protein, meanwhile, introduce coding TEV protease recognition site (ENLYFQG, SEQID) between GST nucleic acid sequence and target gene nucleic acid sequence NO:3) nucleic acid sequence, to facilitat...

Embodiment 2

[0061] The antibacterial activity of embodiment 2 antimicrobial peptide G3

[0062] Taking Gram-negative bacteria E.coli DH5α and Gram-positive bacteria Bacillus subtilis as objects, the antibacterial activity of antimicrobial peptide G3 on the two strains was studied by microdilution method. First, the strains were added to LB liquid medium and beef extract liquid medium for activation, and the activated bacterial liquid was diluted to 2×10 6 CFU / mL used. Add 75 μL of diluted bacterial solution to each well of the 96-well plate, and then add 75 μL of different samples, which are medium blank controls, with a final concentration of 2.5 μM / mL and chemically synthesized G3 at 5 μM / mL as positive controls, with a final concentration of 2.5 Antimicrobial peptide G3 samples prepared in Example 1 at μM / mL and 5 μM / mL were used as experimental groups. Seal the edge of the 96-well plate with ultrapure water, set 4 replicate wells for each group, and the final concentration of bacter...

Embodiment 3

[0063] Application of embodiment 3 antimicrobial peptide G3 in feed

[0064] After freeze-drying the antimicrobial peptide G3 prepared in Example 1, add it to the feed at a rate of 1000 mg per kilogram of feed; wherein, the feed composition in this embodiment is 17% of fish meal, 40% of soybean meal, and 27% of wheat bran , sub-meal 10%, bone meal 3%, allicin 1%, starch polysaccharide 1%-1.5%; feeding medium-length (3-4cm) Penaeus vannamei for 3 months, every 15 days for 3 consecutive days, every day Feed once, and choose to feed in the morning. The results show that compared with the control group without antimicrobial peptide G3, the yield is increased by 15%, and there is no case of death during feeding.

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Abstract

The invention discloses a preparation method of an artificial antibacterial peptide G3, and belongs to the technical field of genetic engineering and molecular biology. A fusion expression method of a glutathione-S-transferase tag and an artificial antibacterial peptide G3 gene is utilized, a TEV protease recognition site is introduced between GST and the artificial antibacterial peptide G3 gene, a recombinant expression vector for expressing GST-TEV cleavage site-G3 fusion protein is constructed, an expression strain is transformed, inducible expression, purification and enzyme digestion are performed, and finally, an enzyme digestion product is purified. The artificial antibacterial peptide G3 is obtained. The preparation method of the artificial antibacterial peptide G3 is high in expression efficiency, simple in separation and purification process, low in production cost and good in stability, and can be applied to large-scale production.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and molecular biology, and in particular relates to a preparation method of artificial antibacterial peptide G3. Background technique [0002] With the abuse of antibiotics, pathogenic microorganisms gradually develop resistance to existing antibiotics, and the problem of microbial resistance has become an urgent problem to be solved all over the world. Antimicrobial peptides have a broad spectrum of anti-pathogenic microorganisms, not only can act on Gram-negative bacteria, Gram-positive bacteria, but also fungi, protozoa and some viruses and tumor cells, and at the same time Normal eukaryotic cells have little effect and have emerged as a potential alternative to traditional antibiotics. Since the discovery of the first insect antimicrobial peptide, more than 3000 antimicrobial peptide molecules have been discovered. However, antimicrobial peptides are present in very small amount...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/70C12N15/62A23K20/147A23K50/80
Inventor 陈翠霞李慧徐海王继乾赵玉荣王栋周兴王玉鸣张再美陈萌琦岳琳路薇郝嘉晨
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)