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Application of glutathione reductase to binding of penicillin antibiotics

A technology of glutathione and reductase, applied in the biological field, can solve problems such as research on bacterial resistance mechanism affecting antibiotic detection, limited antibiotic binding protein, etc.

Inactive Publication Date: 2015-04-29
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of antibiotic-binding proteins discovered so far is limited, which directly affects the detection of antibiotics and the study of bacterial resistance mechanisms.

Method used

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  • Application of glutathione reductase to binding of penicillin antibiotics
  • Application of glutathione reductase to binding of penicillin antibiotics
  • Application of glutathione reductase to binding of penicillin antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cloning of glutathione reductase ETAE_3367 gene

[0025] Glutathione reductase ETAE_3367 gene PCR amplification: According to the gene sequence of Edwardsiella tarda ETAE_3367 published by Gene bank in NCBI, ETAE_3367 gene primers (synthesized by Shanghai Invotrogen Company) were designed for the PCR amplification of Edwardsiella tarda ETAE_3367 increase. The designed primers are: Upstream primer: 5'-GGG GAATTC ATGACTAAACATTATGAC-3', as shown in the sequence SEQ ID NO:2; downstream primer: 5'-CAC AAGCTT TTAACGC ATGGTGACAAA-3', as shown in the sequence SEQ ID NO: 3; the horizontal line shows the enzyme cutting site, and the upstream primer is introduced Eco RI restriction site, downstream primer introduction HindⅢ Restriction sites. Using the Bacterial Genomic DNA Extraction Kit (TIANGEN Company), according to the instructions in the kit, extract the whole genome DNA of Edwardsiella tarda as a template, and use the above primers for PCR amplification...

Embodiment 2

[0031] Example 2 Expression and purification of glutathione reductase ETAE_3367

[0032] Predicted amino acid sequence. DNAssist software was used to analyze the gene ETAE_3367 to obtain its complete open reading frame ORF.

[0033] Prokaryotic expression of recombinants. Pick a single colony of the recombinant plasmid and inoculate it into 1ml of LB liquid medium with kanamycin (50 mg / ml) added at a ratio of 1:500, culture overnight at 37°C until saturated, and inoculate the saturated culture at a 1:100 inoculum to In 5mL of LB medium, culture at 37°C for about 2h to OD 600 It is about 0.6 or so. IPTG was added to the culture to a final concentration of 1 mmol / L, and culture was continued at 37° C. for 3 h. The induced culture was taken out and centrifuged at room temperature for 1 min at high speed to collect the bacteria. At the same time, set up a control group, resuspend the pellet in 100 μL 2×SDS gel loading buffer, and heat at 100°C for 5 minutes. 12000rpm, cent...

Embodiment 3

[0034] Example 3 Purification of glutathione reductase ETAE_3367

[0035] Picking recombinant plasmids and transforming Escherichia coli BL21 (DE3), according to the corresponding vector pET-28a, were cultured overnight at 37°C in LB medium containing 100 μg / mL kanamycin. Immediately pick a single colony, add 5mL LB solution and shake overnight at 37°C, transfer to 200mL liquid LB medium containing antibiotics at a ratio of 1:100, culture at 37°C for about 2.5-3 hours, OD 600 =0.6, add IPTG (final concentration: 1 mmol / L) to the bottle for induction, shake at 200 r / min at 30°C for 3 hours. Centrifuge at 6000g for 10 minutes at 4°C to collect the cells, wash the cells twice with normal saline, and weigh the wet weight of the cells.

[0036] The harvested bacteria were dissolved in buffer D (8M urea; 0.1M NaH 2 PO 4 ; 10mmTris-HCl, pH 8.0) to suspend the bacteria, ultrasonically break, output power 60%, 5s / time, interval 9s, ultrasonication 30min. The sonicated bacteria...

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Abstract

The invention belongs to the field of biotechnology and particularly discloses an application of glutathione reductase to binding of penicillin antibiotics. A sequence of the glutathione reductase is as shown in SEQ ID NO:1. The glutathione reductase ETAE_3367 is a new penicillin antibiotic binding protein and can be effectively applied to the detection of the penicillin antibiotics, the preparation of detection reagents and further research on effects and drug resistance mechanisms of the penicillin antibiotics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of glutathione reductase in combination with penicillin antibiotics. Background technique [0002] Penicillin refers to a class of antibiotics that contain penicillane in the molecule extracted from the culture medium of Penicillium, can destroy the cell wall of bacteria and play a bactericidal effect during the reproduction period of bacterial cells. It is the first antibiotic that can treat human diseases. Penicillin antibiotics are the general term for a large class of antibiotics in β-lactams. They are currently the most widely used class of antibiotics in the pharmaceutical industry. They are widely used in the treatment and prevention of staphylococcus, pneumococcus, streptococcus, actinomycete infections and spirochetes. disease and other diseases. However, long-term intake of a large amount of drugs containing penicillin antibiotics can cause the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12Q1/26
CPCC12N9/0051C12Q1/26C12Y108/01007
Inventor 彭宣宪李惠朱伟聪
Owner SUN YAT SEN UNIV
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