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Antibacterial peptide ID13 as well as preparation method and application thereof

An antibacterial peptide and DNA sequence technology, applied in the field of genetic engineering, can solve the problems of antibacterial activity and cytotoxicity restriction, and achieve the effect of broad application value and market prospect.

Active Publication Date: 2020-04-17
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DLP4 is restricted in clinical application due to its antibacterial activity and cytotoxicity.

Method used

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  • Antibacterial peptide ID13 as well as preparation method and application thereof
  • Antibacterial peptide ID13 as well as preparation method and application thereof
  • Antibacterial peptide ID13 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The design of embodiment 1 antimicrobial peptide ID13

[0049] On the basis of the sequence of Kunming defensin DLP4, a derivative peptide ID13 was designed. The amino acid sequence is shown in SEQ ID NO:1.

Embodiment 2

[0050] The acquisition of embodiment 2 antimicrobial peptide ID13 gene fragment

[0051] 2.1 Optimization and design of antimicrobial peptide ID13 gene expression sequence

[0052] Yeast preferred codon optimization was performed on the coding gene of antibacterial peptide ID13, XhoI restriction site and Kex2 cleavage site were added to the 5' end of the optimized gene sequence, TAA and TAG terminator sequences and XbaI enzyme were added to the 3' end Cutting site, the nucleotide sequence of the resulting gene expression cassette is shown in SEQ ID No:3. The above sequence was completed by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 3

[0053] Example 3 Construction of Yeast Recombinant Expression Vector

[0054] 3.1 The ID13 gene fragment obtained in Example 2 was double digested with XhoI and XbaI endonucleases, and then the purified fragment was recovered. At the same time, the pPICZαA vector (purchased from Invitrogen) was double digested with XhoI and XbaI.

[0055] The double enzyme digestion system is as follows:

[0056]

[0057]

[0058] After adding the sample to the above enzyme digestion system, place it in a PCR instrument at 37°C for 4 hours, and detect it by electrophoresis on a 2% agarose gel. Electrophoresis conditions: 120V, 30min. The digested products were recovered with a DNA product recovery kit. After the ID13 gene and the pPICZαA vector were digested with XbaI and XhoI, the ID13 gene was ligated with the linearized pPICZαA vector with T4 DNA ligase. The connection system is as follows:

[0059]

[0060] Ligation conditions: After loading the above ligation system, connect ...

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Abstract

The invention provides an antibacterial peptide ID13 (with a sequence as shown in SEQ ID No. 1) as well as a preparation method and application thereof. Recombinant expression of ID3 in Pichia pastoris is realized by using a genetic engineering technology. The antibacterial peptide ID13 disclosed by the invention has very good bactericidal activity on gram-positive bacteria such as Staphylococcusaureus, Streptococcus pneumoniae and Streptococcus suis, and has very low hemolytic activity on mouse red blood cells and very low cytotoxicity on mouse-derived macrophages.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an antimicrobial peptide ID13 and its preparation method and application. Background technique [0002] Antimicrobial peptides (AMPs) are endogenous polypeptides produced by multicellular organisms to protect their hosts from pathogenic microorganisms due to their crucial role in constituting the innate immune system. Usually have a wide range of antibacterial activities, including viruses, bacteria, protozoa and fungi (Zasloff et al., 2002); multiple bactericidal target sites, different bactericidal modes from existing antibiotics and broad activities as immunomodulators, so AMPs are considered is one of the strongest candidates for future antibacterial drugs (Ali Adem Bahar et al., 2013). However, natural antimicrobial peptides have disadvantages such as weak antibacterial activity, high synthesis cost, and certain toxicity to eukaryotes. Therefore, how to improve its activ...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/81C12N1/19C12P21/02A61K38/17A61P31/04C12R1/84
CPCC07K14/43563C12N15/815C12P21/02A61P31/04A61K38/00C07K14/43577C07K2319/50C07K14/39C12N1/16
Inventor 王建华李兵滕达毛若雨王秀敏郝娅杨娜
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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