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Miniaturized Staphylococcus aureus polypeptide of against drug-resistance and its uses and preparation method

A staphylococcus, golden yellow technology, applied in the direction of biochemical equipment and methods, chemical equipment and methods, botany equipment and methods, etc., can solve the problems of unfavorable drug safety and high immunogenicity, so as to reduce the toxic effect, Specific antibacterial effect

Active Publication Date: 2009-05-06
畿晋庆堂国际生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the anti-Staphylococcus aureus polypeptide in the above-mentioned patent examples still has some deficiencies in structure and function: the signal domain at the amino terminal of colistin Ia is still retained in the antibacterial polypeptide, resulting in (1) antibacterial polypeptide It has a broad-spectrum antibacterial effect, not only anti-Staphylococcus aureus, but also anti-Escherichia coli-like Gram-negative bacteria; (2) If the molecular weight of the polypeptide is large, the immunogenicity may be greater, which is not good for drug safety

Method used

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  • Miniaturized Staphylococcus aureus polypeptide of against drug-resistance and its uses and preparation method
  • Miniaturized Staphylococcus aureus polypeptide of against drug-resistance and its uses and preparation method
  • Miniaturized Staphylococcus aureus polypeptide of against drug-resistance and its uses and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Construction of a plasmid expressing a miniaturized anti-drug-resistant Staphylococcus aureus polypeptide and preparation of a recombinant miniaturized anti-drug-resistant Staphylococcus aureus polypeptide

[0022] The original plasmid was the pET-15b commercial plasmid loaded with the colistin Ia channel domain and the Immunity protein gene (the size of the plasmid was 6.3kb, purchased from Novagen, and the above-mentioned genes were loaded by the Laboratory of Transplantation and Immunology, West China Hospital, Sichuan University). Oligonucleotide point mutation technology (QuickChange TM Kit, Strategene Company) inserted the gene (SEQ ID NO.2 in the sequence listing) of the coding Staphylococcus aureus pheromone Agr D1 into the I626 site of the colicin Ia channel domain gene, and prepared a part of the antibacterial polypeptide A recombinant plasmid pCHCSACOM1 (such as figure 1 shown). The recombinant plasmid was transfected into E.coli TG1 engineering ...

Embodiment 2

[0045] Example 2 Construction of a plasmid expressing a miniaturized anti-drug-resistant Staphylococcus aureus polypeptide and preparation of a recombinant miniaturized anti-drug-resistant Staphylococcus aureus polypeptide

[0046] The original plasmid was the pET-15b commercial plasmid loaded with the colistin Ia channel domain and the Immunity protein gene (the size of the plasmid was 6.3kb, purchased from Novagen, and the above-mentioned genes were loaded by the Laboratory of Transplantation and Immunology, West China Hospital, Sichuan University). Oligonucleotide point mutation technology (QuickChange TM Kit, Strategene Company) inserts the gene (nucleotide sequence described in SEQ ID NO.2 in the sequence listing) of encoding Staphylococcus aureus pheromone Agr D1 into the D451 site of the colicin Ia channel domain gene to prepare A recombinant plasmid pCHCSACOM2 (such as figure 2 shown). The recombinant plasmid was transfected into the E.coli TG1 engineering bacteria...

Embodiment 3

[0064] Example 3 In vitro inhibition test of penicillin-sensitive Staphylococcus aureus

[0065] Bacteria are American standard strains, ATCC 25923 penicillin-sensitive Staphylococcus aureus, 5 microliters of bacteria liquid (10 8 CFU / ml level of bacteria) add 1% tryptone, 1% NaCl, 0.5% yeast, 0.5% glucose, 1% HK 2 PO 4 A total of 6 groups were prepared in 10ml of culture medium. The first group added 0.2M NaCl+10mM PBS buffer solution (the same amount as the antibacterial polypeptide and control drug liquid added in the experimental group) as a control; the second group added oxacillin, 9 μg / ml, approximately 22nM / ml; The third group added wild-type colistin Ia7μg / ml, about 0.1nM / ml (colicin preservation solution is 0.2MNaCl+10mM PBS buffer); the fourth group added antibacterial engineering polypeptide Ph- disclosed by ZL01128836.1 SA, 7μg / ml, approximately 0.1nM / ml (preservation solution is 0.2M NaCl+10mM PBS buffer); the fifth group added colistin Ia channel domain carbo...

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Abstract

The invention discloses a midget anti drug-tolerance staphylococcus aureus polypeptide comprised of passage structure domain to form ion channel colicine and staphylococcus aureu pheromone, as well as opposite coded nucleotide sequence and recombination plasmid contained said sequence. Wherein, the plupeptide can not induce bacteria to generate trsditional drug-tolerance by the mechnism that it forms directly ion channel on cell film of target cell to kill bacteria. This invention has more single antimicrobial spectrum without effect to Gram-negative bacteria of escherichia coli, shows at least 500 times effect to pheneazonecillin, and can be used as synergist to beta-lactam antibiotics.

Description

technical field [0001] The invention relates to a recombined miniaturized anti-drug-resistant Staphylococcus aureus polypeptide gene, a recombinant plasmid, a polypeptide and an application and preparation method thereof. Background technique [0002] Bacterial infection is a major threat to human life and health. Since the advent of sulfonamides and penicillins, antibiotics have been invented by humans to achieve antibacterial purposes by inhibiting bacterial cell wall synthesis, inhibiting or interfering with bacterial nucleic acid and protein metabolism and synthesis pathways. However, these antibacterial pathways easily induce bacteria to mutate and produce antimicrobial resistance. Therefore, people have been devoting themselves to the development of new antibiotics. [0003] The direct formation of ion channels on the bacterial cell membrane to cause bacterial death is one of the more promising directions for the development of antibiotics. According to this design c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/31A61K38/16A61P31/04C12P21/02
CPCY02A50/30
Inventor 丘小庆
Owner 畿晋庆堂国际生物技术有限公司
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