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Antifungal peptide Drosomycin, preparation method and application thereof

A technology of antifungal peptides and receptor bacteria, applied in antifungal agents, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of low yield of insect antimicrobial peptides, cumbersome extraction steps, and low natural content, etc. Achieve the effects of easy industrial production, high market value, and high expression output

Pending Publication Date: 2019-10-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the natural content of this antifungal peptide in Drosophila melanogaster is very small, and the yield of insect antimicrobial peptides produced by immunization-induced Drosophila is very low, and the extraction steps are cumbersome and costly; direct synthesis is expensive, which limits its development. application bottleneck

Method used

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  • Antifungal peptide Drosomycin, preparation method and application thereof
  • Antifungal peptide Drosomycin, preparation method and application thereof
  • Antifungal peptide Drosomycin, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] 1. Primer design and SUMO-Drosomycin sequence acquisition

[0046] 1. The acquisition of Drosomycin gene SEQ.ID NO.1

[0047]The gene sequence corresponding to the mature Drosomycin peptide is 132bp, as shown in SEQ.ID NO.1, encoding 44 amino acids, the predicted isoelectric point of the protein is 5.45, and the size is 4.9kDa. Drosomycin has a complex structure, with 8 Cys forming 4 pairs of disulfide bonds, which is not suitable for expression in prokaryotic systems. Therefore, according to the above-mentioned characteristics of the peptide, the characteristics of the gene and the expression system, the codons of the Drosomycin gene are first optimized, and the optimized Drosomycin nucleotide sequence is shown in SEQ.ID NO.5. According to the optimized nucleotide sequence, three mutually complementary long primers drs-1, drs-2 and drs-3 were designed respectively, and Drosomycin was directly synthesized by PCR.

[0048] Firstly, the codon-optimized Drosomycin gene w...

Embodiment 2

[0119] For the antimicrobial peptide Drosomycin (Drs) obtained in Example 1, utilize the conventional agar plate hole diffusion method (plate inhibition zone method) to treat Neurospora crassa (N.crassa) and Fusarium oxysporum (F.oxysporum ) was used as the test bacteria to test the antibacterial activity.

[0120] The plate antibacterial test results are as follows: Figure 7 As shown, PBS and a single SUMO protein have no inhibitory effect on the two tested bacteria, while the isolated antimicrobial peptide Drosomycin has an inhibitory effect on the two bacteria when it is above 3 μg, and it shows obvious antibacterial activity at 15 μg .

[0121] Drosomycin and control (PBS) at a concentration of 5 μg / μL were used to act on Neurospora crassa and Fusarium oxysporum respectively. After 10 hours of treatment, the hyphae were fixed with paraformaldehyde, and the JSM-6360LV scanning electron microscope was used to examine the Observed and photographed at 1200 and 3700 times, t...

Embodiment 3

[0123] The CCK-8 method was used to detect the toxicity of the in vitro recombinant protein Drosomycin described in Example 1 to mammalian cell 293T and mouse embryonic fibroblast 3T3-L1, and it was found that it did not affect the proliferation of 293T and 3T3-L1 cells, indicating that the antifungal peptide Drosomycin has no toxic effect on mammalian cells. The result is as Figure 9 As shown, it can be seen that mammalian cells 293-T and 3T3-L1 were treated with 0, 10 μg / mL, 40 μg / mL, and 160 μg / mL of Drosomycin, and 24 hours later, 293-T and 3T3-L1 cells were detected by CCK8 kit As for the proliferation ability, it can be seen from the figure that even if the concentration of Drosomycin is increased to 160 μg / mL, the proliferation ability of 293-T and 3T3-L1 cells is maintained at about 100%. It can be seen that Drosomycin has no toxic effect on mammalian cells 293-T and 3T3-L1.

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Abstract

The invention provides a SUMO-Drosomycin fusion protein and a preparation method thereof, and a preparation method of an antifungal peptide Drosomycin. When expressed in an Escherichia coli expressionsystem, the protein can overcome the problem that most of the target proteins are easily folded in the system, and avoids the formation of non-functional proteins. The protein can be correctly folded, has high antibacterial activity, does not produce drug resistance, and has broad application prospects in preparing antibacterial drugs, sterile foods, and cosmetics, and has high market value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antifungal peptide Drosomycin, a preparation method and an application thereof. Background technique [0002] The extensive use of antibiotics has brought about increasingly prominent problems of bacterial resistance, and may also lead to an imbalance of micro-ecology in the body, resulting in unpredictable hidden dangers of disease. Antimicrobial peptides are the main components of natural immunity. They have the characteristics of high temperature resistance, acid resistance, good water solubility, wide antibacterial spectrum, special antibacterial mechanism, rapid sterilization and no drug resistance, and have become a research hotspot in recent years. [0003] Antibacterial peptides (AMPs), also known as host defense peptides, are ubiquitous in eukaryotic and prokaryotic organisms and participate in the body's natural immunity. Insects are the most widely distributed species ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P21/06C07K19/00C07K14/435A61K38/17A61K8/64A61Q19/00A61P31/10C12R1/19
CPCC12N15/70C07K14/43581A23L33/18A61K8/64A61Q19/00C07K2319/50A61K38/00
Inventor 杨婉莹吴文梅曹阳易辉玉邓小娟黄亚东
Owner SOUTH CHINA AGRI UNIV
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