Design of polypeptides specifically binding to immune protein of pseudomonas aeruginosa type VI secretion system, and validation of antibacterial activity of polypeptides

A secretion system and immune protein technology, which is applied in the field of design of polypeptides specifically binding to the immune protein of Pseudomonas aeruginosa type VI secretion system and the verification of antibacterial activity, which can solve the problems of treatment failure and other problems

Active Publication Date: 2018-06-08
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the emergence of phage antibodies and increased drug resistance have made these treatments ineffective

Method used

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  • Design of polypeptides specifically binding to immune protein of pseudomonas aeruginosa type VI secretion system, and validation of antibacterial activity of polypeptides
  • Design of polypeptides specifically binding to immune protein of pseudomonas aeruginosa type VI secretion system, and validation of antibacterial activity of polypeptides
  • Design of polypeptides specifically binding to immune protein of pseudomonas aeruginosa type VI secretion system, and validation of antibacterial activity of polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Discovery of functional short peptides

[0064] 1. Use petDUET-1 vector as starting plasmid to construct recombinant plasmid. In the recombinant plasmid, the first open reading frame expressed with His N-terminal 6 TplEi mature peptide, the second open reading frame expresses TplE protein.

[0065] 2. Introduce the recombinant plasmid constructed in step 1 into Escherichia coli Rosetta (DE3) to obtain recombinant bacteria.

[0066] 3. Cultivate the recombinant bacteria obtained in step 2, and carry out IPTG induction during the culture process, and then proceed to the following steps in sequence: cell crushing and supernatant, nickel column purification, ion exchange chromatography, Superose 6 molecular sieve chromatography to obtain TplE-TplEi composite Things.

[0067] 4. Take the TplE-TplEi complex obtained in step 3, add subtilisin for in-situ enzymatic digestion and crystallization, and then obtain the crystal and perform structural analysis.

[0068] 5. Accordi...

Embodiment 2

[0071] Example 2. Affinity of functional short peptides to TplEi protein

[0072] The functional short peptide and TplEi protein were combined in vitro to determine the affinity of the functional short peptide. The high affinity of functional short peptides is the first step for the development of new anti-infective drugs.

[0073] 1. Preparation of functional short peptides

[0074] Artificial synthesis of A peptide.

[0075] Artificial synthesis of B peptide.

[0076] 2. Preparation of TplEi protein

[0077] 1. Insert the double-stranded DNA molecule shown in nucleotides 64-1131 of sequence 5 in the sequence table between the NdeI and XhoI restriction sites of pET-28a(+) vector to obtain a recombinant plasmid. After sequencing, the recombinant plasmid has the open reading frame shown in sequence 5 of the sequence list, and the expression N-terminal has His 6 TplEi mature peptide labeled.

[0078] 2. Introduce the recombinant plasmid obtained in step 1 into Escherichia coli Rosetta (DE...

Embodiment 3

[0107] Example 3. Functional short peptides trigger bacterial self-death by activating TplE protein

[0108] 1. Construction of recombinant plasmid

[0109] 1. Insert the double-stranded DNA molecule shown in sequence 6 of the sequence table between the NcoI and XhoI restriction sites of pET-26b(+) vector to obtain the recombinant plasmid pET26b-TplE. After sequencing verification, the recombinant plasmid pET26b-TplE has an open reading frame shown in sequence 6 of the sequence listing. The DNA molecule shown in sequence 6 of the sequence listing encodes the protein shown in sequence 7 of the sequence listing. In sequence 7 of the sequence table, the amino acid residues 1-23 constitute the signal peptide (promoting the secretion of the protein into the periplasmic space), and the amino acid residues 24-592 constitute the TplE protein.

[0110] 2. Insert the double-stranded DNA molecule shown in sequence 8 of the sequence list between the NcoI and XhoI restriction sites of the pET-2...

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Abstract

The invention discloses design of polypeptides specifically binding to immune protein of a pseudomonas aeruginosa type VI secretion system, and validation of antibacterial activity of the polypeptides. The invention first protects a polypeptide, and the polypeptide is shown by a sequence 3 or a sequence 4 in a sequence table. The gene encoding of the polypeptides also falls within the scope of protection of the invention. The invention also protects the application of the polypeptides: binding to the immune protein in the bacteria type VI secretion system; enriching the immune protein in the bacteria type VI secretion system; detecting the immune protein in the bacteria type VI secretion system. The functional short peptides provided by the invention can bond to the immune protein togetherwith effector protein in a competitive way, so that the interaction between TplE and TplEi is destroyed, and the effector protein is further enabled to break down the cell membranes of bacteria and cause the bacteria to die; therefore, the design is a most potent novel antibacterial strategy taking the T6SS effector protein as a target, and a new direction is provided for the treatment of clinically drug-resistant strains.

Description

Technical field [0001] The present invention relates to the design of a polypeptide that specifically binds to Pseudomonas aeruginosa type VI secretion system immune protein (TplEi) and the verification of its antibacterial activity. Specifically, the present invention designs targeted bacterial type VI secretion system effects based on the three-dimensional structure of the protein The protein-immune protein (TplE-TplEi) interacting polypeptide has been found to have antibacterial activity and has the prospect of anti-infective drug development. Background technique [0002] Since the 20th century, infectious diseases caused by pathogenic bacteria have remained a major threat to global public health. The abuse of antibiotics continues to aggravate the emergence of multi-drug-resistant and pan-drug resistant strains, especially in recent years, the discovery of the polymyxin-resistant gene MCR-1 has brought unprecedented challenges to the prevention and control of pathogenic bact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11A61P31/04
CPCC07K14/00
Inventor 崔胜高小攀牟志霞秦博
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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