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Insect antimicrobial peptide Thanatin derivant, producing method and uses of the same

A derivative and antimicrobial peptide technology, which is applied in the field of antimicrobial peptide derivatives, can solve the problems of hemolytic side effects, high cost, and low antibacterial activity, and achieve the effects of simplified purification process, low production cost, and environmental friendliness

Inactive Publication Date: 2008-05-07
沈子龙
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few attempts to directly use natural antimicrobial peptides as antibacterial drugs
The main reasons are: the antibacterial activity of many natural antimicrobial peptides is not high; the cost of chemically synthesizing natural antimicrobial peptides is high, and the yield is low; many antimicrobial peptides have strong hemolytic side effects
For example, melittin extracted from bee venom has strong antibacterial activity, but it also has hemolytic side effects, which limits the application of melittin as an antibacterial drug.

Method used

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  • Insect antimicrobial peptide Thanatin derivant, producing method and uses of the same

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Experimental program
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Effect test

Embodiment 1

[0045] Construction of expression vectors and acquisition of transformants

[0046] The primers (Table 1) were synthesized according to conventional methods, and 6 genes Tq, Ts, Ta, Tqs, Tqa, and Tsa were synthesized by PCR method according to different pairings of primers, separated by agarose electrophoresis, and recovered by gel to obtain the target fragment. According to the instructions, the target fragments were double-digested with BamH I and XhoI respectively, and the corresponding fragments were recovered by the low-melting point agarose method ("Molecular Cloning"). Fragments were ligated to obtain an expression vector containing DNA sequences encoding insect antimicrobial peptide derivatives; the expression vector was transformed into Escherichia coli DH5α competent cells, the transformants were screened and identified, plasmids were extracted by conventional methods, and sent to Shanghai Invitrogen Company for sequencing, and the sequencing results were in line with...

Embodiment 2

[0061] Determination of Antibacterial Activity

[0062] A polypeptide sample solution with a concentration of 6 mg / ml was prepared with sterilized physiological saline. The bacteria used in the test were inoculated in the nutrient broth, cultured at 37°C for 24 hours, diluted 1:105 times with sterile saline before use, and 12 bacterial culture tubes were taken and numbered, and the nutrient meat was added to the first tube Add 1.8ml broth, and add 1.0ml broth to the remaining 11 tubes. Add 0.2ml of polypeptide solution to the first tube, mix well, take out 1.0ml and add it to the second tube, repeat this process, dilute to the 12th tube in turn, add 0.2ml of bacterial solution to each tube, shake gently, place at 37 Cultivate for 24 hours. The minimum concentration of the peptide for sterile growth was used as the minimum sample concentration (MIC) for inhibiting bacterial growth. Table 2 shows the minimum inhibitory concentrations of Thanatin derivatives prepared in Exampl...

Embodiment 3

[0066] Determination of polypeptide hemolytic activity

[0067] Human red blood cells were suspended in phosphate buffer (pH 7.4) to obtain red blood cell suspension (5% v / v). Dissolve the polypeptide in phosphate buffer solution to make about 5mg / ml stock solution, take 14 1.5ml centrifuge tubes, add 1ml polypeptide stock solution to the first centrifuge tube, add 0.5ml phosphate buffer to the other tubes solution, take 0.5ml of peptide stock solution from the first tube and add it to the second tube, mix evenly with a micro mixer, then take 0.5ml solution from the second tube, add it to the third tube and mix evenly, and so on, That is, dilute to the 14th tube sequentially by the doubling dilution method, discard 0.5ml, add 0.5ml of prepared 5% red blood cell suspension to each tube to a final volume of 1.0ml, shake gently, and incubate in a 37°C incubator After 60 min, it was centrifuged at 4000 rpm for 10 minutes, and the supernatant was taken for colorimetry at 414 nm. R...

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Abstract

The invention relates to a group of insect Thanatin ramifications with antibacterial action and the salt which is applicable for medicinal use, as well as a preparation method and the use thereof. Approved by the results of a sterilization experiment, a hemolysis experiment and an irritable partial stimulation experiment, the insect Thanatin ramification has bigger antibacterial activity than Thanatin to colon bacillus, Candida albicans, Klebsiella pneumoniae, and staphylococcus aureus. The hemolysis reaction is not incurred when the concentration is 2.5mg / ml without acute toxicity stimulation reaction.

Description

technical field [0001] The invention relates to antimicrobial peptide derivatives, in particular to a group of antibacterial insect antimicrobial peptide Thanatin derivatives and salts suitable for medicinal use, as well as its preparation method and application. Background technique [0002] The discovery of antibiotics is of great significance in the history of human medicine. Its use has saved countless lives and increased human life expectancy by more than ten years. But with the widespread use of antibiotics, bacteria have gradually adapted and developed resistance to them. Although there are hundreds of antibiotics in clinical use at present, they are all small chemical molecules, and the newly developed antibiotics are generally structural modifications of them, so it is difficult to solve the problem of bacterial resistance to antibiotics. With the emergence of drug-resistant bacteria, the development of new antibiotics against drug-resistant bacteria has become an ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P31/04A61P31/10
Inventor 沈子龙吴国球
Owner 沈子龙
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