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Screening model and application of gram negative bacterium resistant medicament using SecA power pump as target spot

A power pump and model technology, applied in the direction of bacteria, microorganisms, and methods based on microorganisms, can solve problems such as time-consuming, difficult, and expensive

Active Publication Date: 2009-11-18
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the development of new and useful microbial drugs still has great potential, but the research work is becoming more and more difficult, time-consuming and requires more and more funds

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Amplification and expression of Pseudomonas aeruginosa PAO1 SecA gene

[0040] Primer Design for PAO1 secA Gene Amplification

[0041] Oligonucleotide primers were designed based on sequence data published in the Pseudomonas aeruginosa (P. aeruginosa) genome database (www.pseudomonas.com)

[0042] pasecA F1: 5'-TCCCCACGTGTGGATGACCAAGTCCATATG-3' (SEQ ID NO: 1);

[0043] pasecA R1: 5'-GGTGAACCAGGCGGGAAGCTTAGTC--3' (SEQ ID NO: 2),

[0044] The underlined parts in the primers are NdeI and Hind III restriction enzyme cutting sites, respectively.

[0045] PCR amplification of PAO1 SecA gene

[0046] The PCR reaction system includes: 10X reaction buffer, dNTP mixture, pasecA F1 primer, pasecA R1 primer, Triplemaster DNA polymerase, PAO1 genomic DNA and double distilled water without nuclease. The medium volume of the reaction was 50 μl. The conditions of the PCR reaction were: 94°C for 5 minutes, 94°C for 1 minute, 50°C for 1 minute, 72°C for 3.5 minutes, 29 ...

Embodiment 2

[0051] Example 2 Construction of recombinant expression plasmid pET20b / pasecA

[0052] The construction steps of PAO1SecA protein expression plasmid pET20b / pa secA are as follows:

[0053] The pGEM-T / pa secA plasmid and pET20b vector prepared as described in Example 1 were extracted. At 37°C, the two plasmids were digested with NdeI and HindIII, respectively.

[0054] Analysis was performed by agarose gel electrophoresis. use Gel CleanupKit purifies fragments and vectors to be inserted. The amount of insert and vector was estimated by agarose gel electrophoresis.

[0055] The fragment to be inserted was ligated with NdeI and Hind III digested pET20b overnight at 14°C. The molar ratio of fragment to carrier should be about 5:1. Use 10 μl of the ligation mix to transform DH5α competent cells. 200 μl of transformed cells were plated on LB plates containing ampicillin. Incubate overnight at 37°C. Select 10 single colonies and inoculate them into 2ml LB liquid medium cont...

Embodiment 3

[0058] Example 3 Test strain pET20b / pasecA induces expression of Pseudomonas aeruginosa SecA protein in BL21.19

[0059] All transformed cells obtained as described in Example 2 were plated on LB plates containing ampicillin. Incubate overnight at 30°C. Select 10 single colonies for streak purification, pick single colonies and inoculate them into 2ml TAG liquid medium and culture overnight at 30°C. Take 80 μl of the culture and inoculate it into 2 ml of fresh TAG liquid medium until the OD600 reaches 0.8, add 1 mM IPTG, incubate at 25°C for 2 hours, and compare the expression of SecA protein by SDS-PAGE.

[0060] Validation of Complementary Effects of Pseudomonas aeruginosa SecA Protein and Escherichia coli SecA Protein

[0061] Prepare two TAG plates containing 1 mM IPTG and 100 μg / ml ampicillin and two TAG plates containing only 100 μg / ml ampicillin and divide each into 12 equally. The blank control strain BL21.19 / pET20b containing only the blank plasmid and 11 strains o...

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Abstract

The invention relates to a thermosensitive recombinant colon bacillus for efficiently expressing the secA deletion of secA genes of gram negative bacteria such as Pseudomonas aeruginosa, in particularto a novel microbial cell level screening model of a gram negative bacterium resistant medicament using a SecA power pump as a target spot. The screening model utilizes the thermosensitive recombinant colon bacillus efficiently expressing the secA deletion of the secA genes of the Pseudomonas aeruginosa, wild type colon bacilli and the Pseudomonas aeruginosa to carry out microbial cell level gramnegative bacterium resistant medicament screening on a secondary metabolite of a candidate microorganism aiming at the SecA power pump target spot. The screening model can be also used for screeningaiming at a candidate compound.

Description

field of invention [0001] The invention relates to a secA-deleted temperature-sensitive recombinant Escherichia coli, which highly expresses the secA gene of Gram-negative bacteria such as Pseudomonas aeruginosa. The present invention also relates to a novel anti-Gram-negative bacterial drug microbial cell-level screening model targeting the SecA power pump, which utilizes temperature-sensitive E. Escherichia coli and Pseudomonas aeruginosa for microbial cell-level drug screening against Gram-negative bacteria against secondary metabolites of candidate microorganisms against SecA kinetic pump targets. The model can also be used for screening against candidate compounds. Background of the invention [0002] At present, Gram-negative bacteria are still the most important pathogenic bacteria in hospital and community infections, and the problem of drug resistance is increasingly becoming a difficult problem in the treatment of Gram-negative bacteria infections. Drug-resistant...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12Q1/18C12R1/19
Inventor 余利岩赵莉莉司书毅孙承航李秋萍魏玉珍张玉琴刘红宇
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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