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Application of Toll-like receptor ligand protein in resisting bacterial infection

A protein and dual ligand technology, applied in the field of medicine and biology, can solve problems such as adverse side effects and drug resistance, achieve strong innate immune response, and enhance phagocytosis and killing effects.

Pending Publication Date: 2020-01-21
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned shortcoming of prior art, the object of the present invention is to provide the application of a kind of Toll-like receptor ligand protein in anti-bacterial infection, for solving the existing problem of the medicine of anti-bacterial infectious disease in existing treatment strategy. Issues such as drug resistance and adverse side effects

Method used

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  • Application of Toll-like receptor ligand protein in resisting bacterial infection
  • Application of Toll-like receptor ligand protein in resisting bacterial infection
  • Application of Toll-like receptor ligand protein in resisting bacterial infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of recombinant expression vector pET28a(+)-PepO.

[0043] (1) Materials:

[0044] The prokaryotic expression plasmid pET28a(+) was purchased from Novagen, and the Prime Star high-fidelity enzyme, dNTPs, Buffer, and MgCl used in PCR 2 Purchased from Bao Biological Engineering (Dalian) Co., Ltd., PTC-200 PCR instrument is a Perkin Elmer product.

[0045] (2) Design and synthesis of primers:

[0046]Using Streptococcus pneumoniae D39 genomic DNA as a template, refer to its complete sequence (GeneBank number CP000410.2), use Premier5.0 to design primers, and synthesize them by Sangon Bioengineering (Shanghai) Co., Ltd.

[0047] PepO: Upstream primer: 5'-GCCATGGCACGTTATCAAGATGATTTTTAT-3', containing NcoI site;

[0048] Downstream primer: 5'-CCCTCGAGCCAAATAATCACGCGCTCCTCT-3', containing XhoI site.

[0049] (3) PCR amplification of the target gene:

[0050] Amplification of PepO gene, the nucleotide sequence of the amplified PepO gene is shown in SEQ ID NO.1....

Embodiment 2

[0066] Expression, Identification and Purification of Prokaryotic Expression Plasmid pET28a(+)-PepO in Escherichia coli

[0067] (1) The recombinant plasmid pET28a(+)-PepO was transformed into Escherichia coli competent BL21(DE3). Methods and conditions are as described in Example 1. Positive transformants were identified by sequencing and kept for future use.

[0068] (2) IPTG induced the massive expression of PepO recombinant protein.

[0069] Add the preserved pET28a(+)-PepO-BL21(DE3) Escherichia coli into 30mL LB medium, culture at 180rpm at 37°C for 8 hours, then transfer all the bacteria liquid to 500mL LB medium and culture at 180rpm at 37°C for 4h. Then IPTG was added to make the final concentration in the bacterial solution 40 mM, and cultured at 180 rpm at 22° C. overnight.

[0070] (3) Purification of recombinant protein.

[0071] After collecting the bacteria by centrifugation at 12000g at 4°C for 10min, resuspend them in 30mL of binding buffer and sonicate the...

Embodiment 3

[0078] Promotion of macrophage phagocytosis by PepO

[0079] (1) Culture of Streptococcus pneumoniae. Streptococcus pneumoniae D39 strain (NCTC 7466) was obtained from the British National Type Collection (NCTC). Inoculate frozen bacteria on Columbia blood plates at 37°C, 5% CO 2 conditions overnight. Inoculate monoclonal colonies into 10ml C+Y medium at 37°C, 5% CO 2 Cultured until the bacteria were in mid-logarithmic growth phase (OD 600 ) = 0.5.

[0080] (2) Extraction and processing of macrophages. Select 8-week-old C57 / BL6 mice, inject 1 mL of sterilized paraffin oil into the intraperitoneal cavity, kill the mice by neck dislocation after 96 hours, and lavage the abdominal cavity of the mice twice with pre-cooled PBS buffer solution containing 0.1% EDTA-Na , 8mL each time. The lavage fluid was centrifuged at 800 g for 5 min to remove paraffin oil and supernatant, and the erythrocytes were removed with erythrocyte lysate, resuspended in DMEM high-glucose medium, cou...

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Abstract

The invention provides an application of a Toll-like receptor ligand protein in resisting bacterial infection. According to the application, streptococcus pneumoniae endopeptidase O (PepO) is a ligandof a Toll-like receptor 2 and a Toll-like receptor 4, can remarkably enhance phagocytosis and killing effects of macrophages on pathogenic bacteria, and can up-regulate secretion of related cytokinesand chemokines, thereby inducing strong innate immune response and enhancing resistance of respiratory tracts to pathogenic bacteria infection.

Description

technical field [0001] The invention relates to the technical field of medicine and biology, in particular to the application of a Toll-like receptor ligand protein in antibacterial infection, in particular to the application of Streptococcus pneumoniae endopeptidase O in antibacterial infection. Background technique [0002] Antibiotics are the main drugs for the treatment of bacterial infectious diseases. However, due to the widespread and long-term abuse of antibiotics, the outbreak of drug-resistant bacteria and even multi-drug-resistant bacteria has resulted in the current clinical efficacy of antibiotics getting worse and worse. Antibiotic resistance is one of the top ten threats to global health in 2019 listed by the World Health Organization (WHO). It can be seen that bacterial infection is still one of the main problems threatening human health at present, and there is an urgent need to develop new therapeutic drugs. However, the development of new antibiotics is f...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/57C12N15/70A61K39/09A61P31/04
CPCC12N9/52C12N15/70A61K39/092A61P31/04
Inventor 尹一兵李思杰舒钊彻张红张雪梅
Owner CHONGQING MEDICAL UNIVERSITY
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