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Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use

A technology for activating proteins and Staphylococcus aureus, applied in the field of cyclic polypeptides, and can solve problems such as no literature reports

Inactive Publication Date: 2005-01-26
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still controversy about the existence and sequence of RAP, and there is no literature report

Method used

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  • Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use
  • Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use
  • Cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus and its pharmaceutical use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The separation and purification of embodiment 1.RAP (Y)

[0020] Will grow to late logarithmic phase (4.7×10 9 Bacteria / ml) Staphylococcus aureus liquid was centrifuged at 7000×g for 15 minutes to get the supernatant, boiled for 10 minutes, then centrifuged for 15 minutes, and then the supernatant was taken. After concentration ten times with a filter membrane with a molecular weight cut-off of 10kD, 3ml was separated and purified by molecular sieve chromatography. The separation medium was S-300 (100ml, 2.2×26cm), the buffer was 50mM Tris-Cl, pH=8.0, and the target protein peak was collected. Conventional SDS-PAGE polyacrylamide gel electrophoresis was used to detect the purity and observe the molecular weight. The results showed that the obtained protein was a single band with a molecular weight of about 38kD (see attached figure 1 , in the figure 1 represents RAP protein, 2 represents protein standard, 3 represents bovine thrombin (~36kD).

Embodiment 2

[0021] Example 2. Detection of RAP(Y) activity by Northern blot

[0022] The total RNA of Staphylococcus aureus was extracted by conventional methods, and after formaldehyde denaturation electrophoresis, the RNA was transferred to a nylon membrane by semi-dry transfer. After pre-hybridization at 60°C for 1 hour, a labeled RNAIII-specific DNA probe was added. After 16 hours at 60°C, the membrane was washed for autoradiography. In the detection of RAP(Y) activity, the positive control is 0.5ml of late logarithmic growth supernatant (containing high concentration RAP(Y)) concentrated 10 times, adding 4.5ml medium to inoculate early logarithmic growth (5×10 8 Bacteria / ml) Staphylococcus aureus was cultured for 60 minutes; the sample was inoculated with 5 ml of medium and 0.5 mg of purified protein to inoculate the same Staphylococcus aureus and cultivated for 60 minutes; the negative control was inoculated with 5 ml of medium and cultivated for 60 minutes with the same Staphylo...

Embodiment 3

[0024] Example 3. Screening of RAP(Y) binding peptides

[0025] First, 100 μl of purified RAP(Y) protein was used to coat the enzyme-linked plate, and placed at 4° C. overnight. After blocking with 2% gelatin for 1 h, add the phage peptide library, incubate at room temperature for 1 h, wash the non-specifically bound phage with TBST (50 mmol / L Tris-HCl, 0.1% TWEEN20, pH 7.5), and then wash with 0.2 mmol / L glycine- HCl pH 2.2 was used to elute the specifically bound phage, and the eluate was neutralized with 1 mmol / L Tris-HCl pH 9.0. According to the method provided by the kit, the titer of the phage in the eluate was determined, and the titer of the eluted phage of the uncoated target protein was used as a control to determine the input-output ratio. At the same time, the eluted phages combined with RAP were amplified and their titers were determined for the next round of screening. After 3 rounds of screening, the input and output of the assay had been significantly impro...

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Abstract

The invention relates to a group of cyclo-polypeptides capable of combining with autocrine RNAIII activator protein of gold staphylococcus, Steroidal C#-[1]-X#-[1]-H#-[1]-A#-[2]-H#-[1]-A#-[2]-C#-[1], wherein C is natural L-type aminothiopropionic acid residue or its D-type isomer, X is any one of the four natural L-type amino acid residue or its D-type isomers, i.e. glutamine residue (Q), glutacid residue (E), aspartate residue (D), asparagines residue (N), H is L-type histidine residue or its D-type isomer, A is natural L-type aromatic residue or its D-type isomer. The polypeptides can be applied in preparing staphylococcus aureus resistant medicament.

Description

technical field [0001] The invention relates to a group of cyclic polypeptides, in particular to cyclic polypeptides capable of binding to the self-secreted RNAIII activation protein of Staphylococcus aureus, and the binding peptide can specifically inhibit the toxin production of Staphylococcus aureus. The present invention also relates to the application of these cyclic polypeptides in the field of medicine. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is a common Gram-positive pathogen, and it is one of the main microorganisms that cause fatal diseases such as burns and war wound infections, pneumonia, endocarditis, sepsis, and toxic shock. one. The number of people infected with Staphylococcus aureus in hospitals alone exceeds millions every year. At present, the combined use of antibiotics is often used in clinical treatment of Staphylococcus aureus, but the effect is not ideal. Because Staphylococcus aureus is prone to drug resistance ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P31/04C07K7/06
CPCC07K7/06A61P31/04
Inventor 邵宁生杨光柳川高亚萍董洁丁红梅沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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