Bombina orientalis polypeptide, and preparation method and application thereof

A technology of bombes and polypeptides, applied in the field of oriental bombesin polypeptides and its preparation, can solve the problems of bacterial resistance to antibiotics, achieve low immunogenicity, solve drug residues and drug resistance of pathogenic bacteria in humans and animals, and inhibit inflammation The effect of the reaction

Inactive Publication Date: 2016-08-10
INFINITUS (CHINA) CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The development of antimicrobial peptide products into new products that replace traditional anti

Method used

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  • Bombina orientalis polypeptide, and preparation method and application thereof
  • Bombina orientalis polypeptide, and preparation method and application thereof
  • Bombina orientalis polypeptide, and preparation method and application thereof

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preparation example Construction

[0030] The present invention also provides a preparation method of the Bombins orientalis polypeptide ABP27, comprising the following steps:

[0031] 1) Solid-phase synthesis of ABP27-amino resin;

[0032] 2) Cleavage of ABP27-amino resin to obtain ABP27 crude peptide;

[0033] 3) The crude ABP27 peptide was dissolved in 1% acetic acid solution, and separated by gel column chromatography to obtain the refined ABP27 peptide.

[0034] Wherein, step 1) of the preparation method of the present invention adopts the Fmoc / tBu synthesis strategy, uses amino resin with a suitable degree of substitution as a carrier, and solid-phase synthesizes amino acids coupled with Fmoc protecting groups one by one to obtain ABP27-amino resin.

[0035] Preferably, the solid-phase synthesis is Fmoc-Arg (Pbf)-OH and amino resin to react to synthesize Fmoc-Arg (Pbf)-amino resin, and then adopt other amino acids of coupling Fmoc protecting group to obtain ABP27 in a coupling manner one by one - Amino ...

Embodiment 1

[0064] Embodiment 1: Preparation of polypeptide ABP27

[0065] 1. Preparation of Fmoc-Arg(pbf)-amino resin

[0066] Add 10.03g of Fmoc-Rink Amide with a substitution degree of 0.84mmol / g to the solid phase reactor, add DMF to soak, make it fully swell for 30min, drain it, add 20% piperidine / DMF solution to remove the Fmoc protection group 2 times, the first reaction was 5 minutes, the second reaction was 15 minutes, and washed with DMF, DCM and methanol. Take 11.1g of Fmoc-Arg(Pbf)-OH, 2.24g of HOBt, 6.14g of HBTU dissolved in DMF and add to the reactor to equilibrate for 5min, add 6.0mL of DIPEA to react at room temperature for 1 hour, drain and wash with DMF, DCM and methanol for 3 The second time, dry and weigh to obtain 17.12g of Fmoc-Arg(pbf)-Rink Amide resin, and the detection degree of substitution is 0.67mmol / g.

[0067] 2. Preparation of ABP27 amino resin

[0068] Put the above resin in a peptide bottle, add 20% piperidine / DMF solution to remove the Fmoc protecting...

Embodiment 2

[0073] Embodiment 2: the mensuration of ABP27 antimicrobial spectrum

[0074]Single colonies of Staphylococcus aureus, Escherichia coli DH5α, Bacillus acnes, zoopathogenic Escherichia coli, Candida albicans, and Saccharomyces saccharomyces that were identified as positive by PCR were picked from the YPD plate and inoculated in 100ml BMGY medium, and cultured with shaking at 30°C until OD600 2-6, remove the supernatant, add 20ml of BMMY medium to resuspend the bacteria, shake culture at 30°C for 96 hours, take 1mL samples in Eppendorff tubes every 24 hours, and take the supernatant for antibacterial activity detection.

[0075] Heat and melt the agar medium in a water bath, cool it to about 50°C, draw 60 μL of bioassay bacteria (OD600=0.3) by aseptic technique, add it to 20mL agar medium, mix quickly, and pour it into a 9cm diameter container. In a sterile flat dish, place horizontally and wait for solidification. A round hole with a diameter of 2.7 mm was punched on the agar,...

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Abstract

The invention relates to the field of polypeptide drugs, specifically to a Bombina orientalis polypeptide, and a preparation method and application thereof. The Bombina orientalis polypeptide ABP27 has an amino acid sequence as shown in SEQ ID No. 1. Experimental results show that the ABP27 has good antibacterial activity to Staphylococcus aureus, Escherichia coli DH5alpha, Bacillus acnes, animal pathogenic Escherichia coli, Candida albicans and Saccharomyces sake. Moreover, the ABP27 has good corrosion resistance and can used as an antiseptic substitute. The ABP27 is capable of inhibiting inflammatory response of Propionibacterium acnes. The Bombina orientalis polypeptide ABP27 provided by the invention can be used for preparing antibacterial drugs or cosmetics, has a wide application scope and produces good economic and social effect.

Description

technical field [0001] The invention relates to the field of polypeptide drugs, in particular to a Bombina orientalis polypeptide and its preparation method and application. Background technique [0002] In recent years, with the abuse of antibiotics, there are more and more drug-resistant bacteria, and bacterial drug resistance is becoming more and more serious. According to data, 95% of Staphylococcus aureus isolated clinically in the United States are resistant to penicillin, and more than 50% are resistant to methicillin (FrindkinSK, Gaynes RP.Antimicrobial resistance in intensive care units.Clin ChestMed.1999,20 (2):303-316). In 2012, my country's clinical testing found that the average detection rates of methicillin-resistant strains (MRSA and MRCNS) in Staphylococcus aureus and coagulase-negative staphylococcus were 47.9% and 77.1% respectively (2012 China CHINET Bacterial Resistance Surveillance Chin J Infect Chemother, 2013, 13(5): 321-330). This situation serious...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12C07K1/16C07K1/06C07K1/04A61K8/64A61K38/17A61P31/04A61P31/10
CPCA61K8/64A61K38/00C07K14/463Y02A50/30
Inventor 华洋林梁东李莉楠唐健
Owner INFINITUS (CHINA) CO LTD
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