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A kind of novel antibiotic containing antibody mimetic and its preparation method and application

An antibody mimetic, antibiotic technology, applied in the field of biomedicine, can solve problems such as drug resistance and bacterial mutation

Active Publication Date: 2012-02-15
PHEROMONICIN BIOTECHNOLOGY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these antibacterial methods can easily induce bacterial mutations and produce drug resistance

Method used

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  • A kind of novel antibiotic containing antibody mimetic and its preparation method and application
  • A kind of novel antibiotic containing antibody mimetic and its preparation method and application
  • A kind of novel antibiotic containing antibody mimetic and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] [Example 1] Construction of plasmids expressing novel antibiotics and preparation of novel antibiotics

[0050] The original plasmid is pSELECT loaded with colicin and immunity protein genes TM -1 plasmid (8.3kb). Double-stranded oligonucleotide point mutation technology (QuickChange TM Kit, Strategene Company) will encode the antibody mimetic gene, such as the nucleotide sequence described in Seq ID No.1 and Seq ID No.3 respectively inserted on the 626 position of the colicin allosteric polypeptide gene, prepared a novel Antibiotic mutant plasmids pBHC-PorA1, pBHC-PorA2 (such as Figure 1-Figure 2 shown). The mutant plasmid was transfected into E.coli BL-21 engineering bacteria to prepare new antibiotics.

[0051] The mutation program was carried out according to the Strategene QuickChange SiteDirected Mutagenesis Kit (catalog#200518) kit manual: namely

[0052] 1. Prepare the point mutation reaction:

[0053] 5ul 10X buffer

[0054] 2ul (10ng) original plasmid...

Embodiment 2

[0103] [Example 2] The inhibitory effect of novel antibiotics on Neisseria meningitidis

[0104] The bacterium is the 29332 meningococcal strain of the China Culture Collection Center, and the bacterial solution is 2 microliters (10 5 CFU / ml) was added to 10 ml of rabbit blood chocolate culture solution (50 mg of beef extract, 100 mg of tryptone, KH 2 PO 4 30mg, NaCl 50mg, defibrillated rabbit blood 0.5-0.8ml), prepare 5 groups in total, add 0.3M NaCl+50mM boric acid buffer solution (that is, the blank preservation solution of new antibiotics, the amount is the same as the new antibiotics added in the experimental group) The amount of liquid is the same) as a control, the second group was added with 5 μg / ml ampicillin, the third group was added with 5 μg / ml PMC-AM1, the fourth group was added with 5 μg / ml PMC-AM2, and the fifth group was added with 10 μg / ml PMC-AM1.

[0105] The above-mentioned groups of liquids were respectively placed in 100 ml Erlenmeyer flasks, grown at ...

Embodiment 3

[0106] [Example 3] Comparison of the minimum inhibitory concentration of new antibiotics against multi-drug resistant Pseudomonas aeruginosa and the minimum inhibitory concentration of currently used antibiotics.

[0107] The minimum inhibitory concentration (MIC) of new antibiotics was determined by agar double dilution method. Bacteria were inoculated on the surface of agar plates containing different drug concentrations with a multi-point inoculation instrument (DeneleyA400), and the bacterial content of each point was 10 5 CFU / ml, incubate at 37°C for 18-24 hours to observe the results, and take the lowest concentration of the drug contained in the plate culture medium without bacteria as the minimum inhibitory concentration (MIC value) of the drug to the bacteria.

[0108] The bacterial species used is multi-drug resistant Pseudomonas aeruginosa (clinical isolate 13578 of West China Hospital), and the culture medium is MH medium (every 100 milliliters: 500 mg of beef extr...

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Abstract

The present invention belongs to field of biology and medicine, and especially relates to a novel antibiotic comprising a mimetic antibody, its preparation methods and uses thereof. The novel antibiotic comprising comprising an antibody mimetic and a colicin,or comprising an antibody mimetic and a channel-forming domain of a colicin, the antibody mimetic covalently bonded to the carboxyl end of the polypeptide of the colicin or the channel-forming domain of the colicin, wherein the colicin is selected from a group consisting of colicin E1, Ia, Ib, A, B, N; wherein said antibody mimetic being yielded by fusing two complementarity determining regions (CDRs), V H CDR 1 ) and V L CDR through a cognate framework region (V H FR 2 ) of an immunoglobulin; wherein the immunoglobulin specifically recognizes bacterium porins. Its antibacterial ability is a thousandfold powerful than normal antibiotics. Due to its unique action mechanism, drug resistance resulted in mutation can hardly be acquired by pathogenic bacteria. And the antibiotic will not hurt normal human cells when it kills pathogenic bacteria. Therefore, it can be used for manufacturing antibacterial medicament of killing Diplococcus intracellularis, vancomycin-resistant enterococci, Methicillin-resistant Staphylococcus aureus, or multidrug resistant Pseudomonas aeruginosa.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a novel antibiotic containing an antibody mimic and its preparation method and application. Background technique [0002] Since penicillin and other antibiotics were put into use in 1944, not only meningococcus, but also many other life-threatening pathogenic bacteria, such as Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis have developed resistance to it sex. 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] Currently commonly used 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 bact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/63A61K47/48A61K38/16C12P21/02A61P31/04C12N15/62
CPCC07K16/1271C07K16/1228C07K16/1217C07K2318/00A61K38/00C07K16/1214C07K16/12A61K47/48484A61K47/48507A61K47/6829A61K47/6835A61P31/04
Inventor 丘小庆
Owner PHEROMONICIN BIOTECHNOLOGY LTD
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