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Antimicrobial peptide cecropin A mutant and coding gene, preparation method and application thereof

A technology of peptide cecropin and mutants, applied in the fields of genetic engineering and biopharmaceuticals, can solve the problems of weak antibacterial performance, achieve strong antibacterial performance, good non-hemolytic properties, and good thermal stability

Active Publication Date: 2018-12-18
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the natural cecropin A antibacterial peptide has better antibacterial performance against Gram-negative bacteria, compared with other antibacterial peptides, there is still much room for improvement in its antibacterial activity
In addition, its antibacterial properties against Gram-positive bacteria are weak, so it is an important direction for the study of cecropin A antibacterial peptides to design cecropin A mutant peptides with stronger antibacterial properties through molecular design and directional transformation

Method used

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  • Antimicrobial peptide cecropin A mutant and coding gene, preparation method and application thereof
  • Antimicrobial peptide cecropin A mutant and coding gene, preparation method and application thereof
  • Antimicrobial peptide cecropin A mutant and coding gene, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: the gene design of antimicrobial peptide PEW300

[0044] (1) According to the amino acid sequence composition, charge and hydrophobicity analysis of the natural antimicrobial peptide cecropin A, amino acids in non-conserved regions were selected for amino acid mutation. In order to improve the positive charge of the N-terminus and the hydrophobicity of the C-terminus of the antimicrobial peptide, the Glu at the 9th position was replaced with His, the Asp at the 17th position was replaced with Lys, and the Thr at the 33rd position was replaced with Ala. The amino acid sequence composition of the finally designed antimicrobial peptide is shown in SEQ ID No:3. The physical and chemical properties of the natural antimicrobial peptide cecropin A and the mutant peptide PEW300 are shown in Table 1.

[0045] (2) Through the homology modeling of the mutant peptide PEW300, and the analysis of the electron cloud and hydrophobicity analysis at the mutation site, it c...

Embodiment 2

[0046] Example 2: Construction of recombinant expression vector pET-pew300

[0047] (1) According to the gene sequence of the self-aggregating short peptide ELK16, the self-cleaving short peptide Mxe GyrA and the linking peptide PT linker (PTPPTTPTPPTTPTPTP), the corresponding DNA sequence Mxe-PTlinker-ELK16 was synthesized at Gene Synthesis Company (Shanghai Sangong), and synthesized Two enzyme cleavage sites, Nde I and Xho I, were introduced into the upstream and downstream of the Mxe-PT linker-ELK16 gene fragment respectively. Then, the pET30a(+) plasmid and the Mxe-PT linker-ELK16 gene fragment were double-digested respectively, and connected to form the plasmid pET-Mxe-PTlinker-ELK16.

[0048] (2) According to the gene sequence of the antimicrobial peptide, its DNA sequence PEW300 was synthesized in Gene Synthesis Company (Shanghai Sangong), and the PEW300 gene was synthesized into pUC57 plasmid to form pUC57-PEW300 plasmid. Design the following primers (PEW300-F / PEW300-...

Embodiment 3

[0066] Example 3: Construction of engineering bacteria expressing PEW300-ELK16 fusion protein in Escherichia coli

[0067] The recombinant expression vector pET-PEW300 constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) competent cells by chemical transformation, and then the bacterial solution was applied to the LB solid plate containing 50ug / ml kanamycin Incubate overnight at 37°C. Among them, BL21 (DE3) competent cells were purchased from Zhenzhi Biotechnology Co., Ltd. The BL21(DE3) engineering bacteria containing the recombinant plasmid pET-PEW300 were obtained.

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Abstract

The invention discloses an antimicrobial peptide cecropin A mutant and a coding gene, a preparation method and application thereof. The amino acid sequence of the mutant is as shown in SEQ ID No: 3. The nucleotide sequence of a gene for encoding the above mutant is as shown in SEQ ID No: 4. Molecular cloning is carried out according to the gene sequence of the antimicrobial peptide mutant PEW300 at the design site, and the gene was cloned into a recombinant expression vector; and through fusion expression with a self-aggregation short-peptide ELK16, high-efficiency expression of the mutant PEW300 is realized. Then, bacteriostasis performance test of 9 indicator bacteria is carried out on the mutant peptide PEW300, and the results show that bacteriostasis performance of the antimicrobial peptide PEW300 is greatly improved and the antimicrobial peptide PEW300 has strong pH and heat stability.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and biopharmaceuticals, and in particular relates to the preparation and application of an antimicrobial peptide Cecropin A mutant. Background technique [0002] For a long time, with the overuse of antibiotics, the phenomenon of bacterial resistance to antibiotics has become increasingly serious. With the general understanding of bacterial resistance to antibiotics, the research and development of new antibacterial drugs is imminent. Because antimicrobial peptides have the advantages of broad antibacterial spectrum, unique mechanism of action, not easy to produce drug resistance, thermal stability and good water solubility, it has quickly become a hot spot for scientists at home and abroad to research and develop new antibacterial drugs, and it is expected to become the most potential alternative to antibiotics. One of the antibacterial drugs, has broad application prospects. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P31/04
CPCA61K38/00A61P31/04C07K14/43563C12N15/70
Inventor 王菊芳王蒙马毅傅宏鑫
Owner SOUTH CHINA UNIV OF TECH
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