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Composition for preventing or treating staphylococcus aureus infection

A staphylococcus, composition technology, applied in the direction of antibacterial drugs, antifungal agents, medical preparations containing active ingredients, etc., can solve difficult identification, difficulty in prevention and treatment of infectious diseases, separation/purification of difficult materials, etc. question

Inactive Publication Date: 2017-02-15
GREEN CROSS CORP THE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ligand materials of bacteria recognized by in vivo defense proteins in the host have not been clearly identified, thus presenting difficulties in the prevention and treatment of infectious diseases caused by pathogens
[0007] In particular, ligands that are cell wall components of Staphylococcus aureus (such as WTA, LTA, PGN, CP, and lipoproteins, etc.) are mainly sugar polymers with complex structures, and are purified together with other materials, so it is difficult as a single material separation / purification
Additionally, since multiple types of cell wall components are exposed, it is not easy to identify which components act as ligands for the host's in vivo defense proteins

Method used

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  • Composition for preventing or treating staphylococcus aureus infection
  • Composition for preventing or treating staphylococcus aureus infection
  • Composition for preventing or treating staphylococcus aureus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Embodiment 1: Preparation is used to obtain the bacterial strain of WTA derivative

[0126] For the isolation of WTA-PGN, S. aureus T384 strain (RN4220ΔlgtΔoatA double mutant) was prepared according to the method described in the reference [Kazue Takahashi et al., Plos One 8: e69739, 2013].

[0127] Briefly, bacteriophage 80 was used as a medium by transforming the T363 strain with lipoprotein diacylglycerol transferase (lgt) deletion (Nakayama M et al., Journal of Immunology 189:5903-591, 2012) and with erythromycin resistance. And T0003 strain (Park KH et al., Journal of Biological Chemistry 285, 27167-27175, 2010) with O-acetyltransferase (oatA) gene deletion was used to prepare Staphylococcus aureus T384 strain. Due to the deletion of the lgt gene, this strain can be used to isolate WTA, WTA-PGN and PGN without lipoprotein contamination, and due to the absence of an acetyl group at the oxygen at position 6 of the PGN MurNac residue due to the deletion of the oatA ge...

Embodiment 2

[0128] Example 2: Isolation and purification of soluble WTA-PGN

[0129] Insoluble WTA-PGN was obtained from the Δlgt / ΔoatA mutant strain prepared in Example 1, and soluble WTA was isolated from the insoluble WTA-PGN and purified (see figure 2 ).

[0130] Isolation and purification of insoluble WTA-PGN derivatives

[0131] Insoluble WTA-PGN was isolated and purified by modifying the method described in references [Park KH et al., Journal of Biological Chemistry 285, 27167-27175, 2010; Jung DJ et al., Journal of Immunology 2012, 189: 4951-4959, 2012].

[0132] Specifically, the ΔlgtΔoatA mutant strain of Example 1 was cultured in an incubator, and the obtained bacterial cells were collected. Suspend the recovered bacterial cells (10 mL) in 20 mM citrate buffer (pH 4.5; 30 mL), dilute 50 μL of it 400-fold, and adjust it to OD using a spectrophotometer 600nm is 0.8. Then, in order to remove the 20 mM citrate buffer (pH 4.7), the resultant was centrifuged at 10,000 rpm for...

Embodiment 3

[0151] Example 3: Purification of Soluble WTA

[0152] For purification of WTA, insoluble WTA-PGN (80 mg) was suspended in 20 mM citrate buffer (pH 4.5; 19 mL), and trichloroacetic acid (100 mg / mL; 1 mL) was added to a final concentration of 5 mg / mL. The suspension was reacted in an incubator at 30°C for 12 hours while stirring at 180 rpm, and then centrifuged at 10,000 rpm for 10 minutes at 4°C. Transfer the supernatant to a 50 mL tube, precipitate with acetone for 1 h, and centrifuge at 15,000 rpm for 25 min at 4 °C. The resulting pellet was transferred to a 1.5 mL microcentrifuge tube to remove acetone and suspended in 20 mM Tris-HCl buffer (pH 7.0; 1 mL).

[0153] Then, the resultant was subjected to HPLC using a Hitrap-Q column. All lines and columns were washed with A buffer which was 20 mM Tris-HCl (pH 7.0) and the detector was set to detect and measure absorbance at 220 nm at a sensitivity of 1 and equilibrated. Filter the sample to be loaded with a 0.45 μm filter a...

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Abstract

The present invention relates to: a composition containing, as an active ingredient, wall teichoic acid (WTA)-attached peptidoglycan (PGN); a method for preventing or treating Staphylococcus aureus infection by using the composition; and a method for preparing soluble wall teichoic acid-attached peptidoglycan (WTA-PGN), which can be used as an active ingredient in the composition. The composition of the present invention can be utilized for the prevention or treatment of Staphylococcus aureus infection by: opsonophagocytosis caused by an antigen-antibody reaction; and neutrophil-mediated phagocytosis caused by T cell activation during the early stage of infection.

Description

technical field [0001] The present invention relates to the composition that is used for preventing or treating Staphylococcus aureus (Staphylococcus aureus) infectious disease, more specifically relates to the peptidoglycan (wall teichoic acid-attached peptidoglycan, hereinafter referred to as WTA- PGN) as an active ingredient, a method of using the composition for preventing or treating Staphylococcus aureus infectious diseases, and a method for preparing soluble WTA-PGN that can be used as an active ingredient in the composition. Background technique [0002] Staphylococcus aureus can cause serious infections in human skin, soft tissues and bloodstream (Lowy FD, The New England journal of medicine, 339:520-532, 1998). In addition, S. aureus can become a methicillin-resistant Staphylococcus aureus (MRSA) strain that is resistant to methicillin, a beta-lactam antibiotic, and such MRSA infections are more difficult treatment and has a poor prognosis, thus becoming a big soc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/14A61K38/17A61P31/04A61P31/10
CPCA61K38/17A61K38/14A61P31/04A61P31/10C07K9/00C12P21/005
Inventor 李福律李民姿李宗虎成慜荣安东浩高桥和枝黑川健儿
Owner GREEN CROSS CORP THE
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