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Novel antibiotic and nucleotide sequence, preparation method and application thereof

A technology of nucleotide sequences and antibiotics, applied in the field of biomedicine, to achieve good bactericidal effect, reduce intake, and good in vivo protection effect

Active Publication Date: 2010-02-10
美国信息菌素生物科技公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the specific pheromone of pathogenic bacteria can be used as an inducer to change the targeting of colistin, it should be an ideal antibiotic development direction. At present, the inventors of the present invention have obtained the combination of E. The patent right for the expression of antifungal polypeptides by linking coccoid pheromone genes, the patent number is ZL200510020219.9, but because there are some structural domains in the colicin peptide chain that may cause hypersensitivity, it is necessary to improve

Method used

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  • Novel antibiotic and nucleotide sequence, preparation method and application thereof
  • Novel antibiotic and nucleotide sequence, preparation method and application thereof
  • Novel antibiotic and nucleotide sequence, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] [Example 1] Construction of plasmids expressing novel antibiotics and preparation of novel antibiotics

[0058] The original plasmid is pSELECT loaded with colistin allosteric polypeptide Ia' and immunity protein gene TM -1 plasmid (8.3kb) (purchased from Promega). Double-stranded oligonucleotide point mutation technology (QuickChange TM Kit, Strategene Company) will encode the staphylococcal pheromone gene sequence as shown in: SEQ ID NO 1, 3, 5, 7, 9, respectively inserted into the 626th amino acid position of the colicin allosteric polypeptide Ia' gene to obtain A series of recombinant plasmids for preparing new antibiotics, pBHC-SA1, pBHC-SA2, pBHC-SA3pBHC-SA4, pBHC-SE, such as Figure 1 to Figure 5 Shown, and the gene sequence of Staphylococcus aureus pheromone AgrDI as shown in SEQ ID NO 1 is connected before the No. 1 amino acid position of the allosteric polypeptide Ia' gene to obtain recombinant plasmid pBHC-PA as shown in Figure 6 shown. The recombinant ...

Embodiment 2

[0106] [Example 2] Inhibition of novel antibiotics to penicillin-sensitive Staphylococcus aureus

[0107] Bacteria are American standard strains, ATCC 25923 penicillin-sensitive Staphylococcus aureus, 2 microliters of bacteria solution (10 5 CFU / ml) add 1% tryptone, 1% NaCl, 0.5% yeast, 0.5% glucose, 0.1% K 2 HPO 4 In 10 milliliters of the culture fluid of this method, prepare 6 groups altogether, the first group adds 0.3M NaCl+50mM boric acid buffer solution (namely the blank preservation of protein produced by the BL-21 engineered bacterium of novel antibiotic, wild-type colistin Ia and no plasmid) liquid, the amount is the same as the amount of new antibiotic liquid added in the experimental group) as a contrast, the second group added 100ng / ml penicillin G sodium, the third group added 100ng / ml wild-type colicin Ia, and the fourth group added 100ng / ml For the protein produced by BL-21 engineering bacteria without plasmid, the fifth group added 100ng / ml colicin Ia carboxy...

Embodiment 3

[0109] [Example 3] Transmission electron microscope observation of the bactericidal effect of the new antibiotic PMC-SA1 on methicillin-resistant Staphylococcus aureus (ATCC BAA-42) (stained with 1% phosphotungstic acid, magnified 25,000 times).

[0110] Culture medium: 1% tryptone, 1% NaCl, 0.5% yeast, 0.5% glucose, 0.1% K 2 HPO 4 .

[0111] Con: For the control group, bacteria were added to the aforementioned culture solution and appropriate amount of 0.3M NaCl+50mM boric acid buffer at 200rpm, and after 2 hours of growth at 37°C, the morphology of the bacteria was still normal;

[0112] Oxa: After the bacteria were added to the above culture solution and oxacillin 500ug / ml at 200rpm and grown at 37°C for 1.5 hours, the color and shape of the bacteria changed, but the bacteria remained intact;

[0113] PMC: After the bacteria were added to the aforementioned culture solution and treated with PMC-SA1 10ug / ml for half an hour, the bacteria burst and the contents leaked.

[...

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Abstract

The invention relates to a novel antibiotic and a nucleotide sequence, a preparation method and an application thereof, belonging to the biopharmaceutical field. The novel antibiotic comprises colicinallosteric polypeptide and staphylococcus pheromone peptide chain which are capable of specifically killing pathogenic bacteria combined by the specificity of the antibiotic without damaging the normal cells of human body and are linearly linked with each other to generate novel antibiotics resisting staphylococcus aureus, staphylococcus epidermidis and pseudomonas aeruginosa, has the antisepticeffect as thousand times as the conventional antibiotics, difficult generation of drug resistance, and safer use through overcoming the shortcoming of causing the hypersensitivity of human body of wild colicin.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a novel antibiotic and its nucleotide sequence, preparation method and application. Background technique [0002] Since penicillin and other antibiotics were put into use in 1944, bacteria, especially life-threatening pathogenic bacteria, such as Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, have developed resistance to them. According to relevant reports published by the U.S. Centers for Disease Control (CDC) over the years, it is predicted that these antibiotics may be completely ineffective in another 10 to 20 years. [0003] The above-mentioned antibiotics mainly achieve antibacterial purposes by inhibiting cell wall synthesis, inhibiting or interfering with bacterial nucleic acid and protein metabolism and synthesis pathways. However, these antibacterial methods are easy to induce bacterial mutations and drug resistance. ...

Claims

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Application Information

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IPC IPC(8): C07K14/245C12N15/31A61K38/16A61P31/04C07K19/00C12N15/62C12N15/63C12P21/02C12R1/19
Inventor 丘小庆
Owner 美国信息菌素生物科技公司
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