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Organism identification method based on membrane-bound complex

A complex, membrane-bound technology, applied in the field of cell identification, can solve the problems of seldom application in the pharmaceutical field, difficult to control the reaction, difficult to scale up the process, etc., and achieve the effect of mild process, short time-consuming, high repeatability and accuracy.

Inactive Publication Date: 2019-08-13
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effect of this patented technology is that it allows quicker and simpler ways to identify or distinguish harmful microorganisms by measuring their specific properties such as color changes caused when certain compounds react with proteins inside them (fluorochromism). These techniques also allow researchers to study how these modifications affect biological functions like metabolic activity and immune response.

Problems solved by technology

This patented problem addressed in this patents relates to identifying drug targets from complex mixtures containing many different components like lipids or other small organisms with varying concentrations. Current techniques involve multiple stages involving tedious procedures, costly equipment, and long periods of operation due to their limitations. Therefore there is a desire to develop simpler yet more efficient ways to identify drug targets without requiring extensive analysis.

Method used

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  • Organism identification method based on membrane-bound complex
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  • Organism identification method based on membrane-bound complex

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Design and expression of transmembrane binding proteins

[0023] Ib-AMP4 is a plant antimicrobial peptide, which was first discovered in Impatiens balsamina plants and is a broad-spectrum antimicrobial peptide. GFP is green fluorescent protein, which is often used for molecular tracking. The amount of GFP can be judged by the fluorescence intensity in the solution. Ib-AMP4-GFP, GFP-Ib-AMP4, thanatin-GFP and Ib-AMP4-GFP-RGD four fusion proteins are prepared in the same way, all by designing corresponding nucleic acid sequences and applying genetic engineering methods to construct expression plasmids And express and purify in Escherichia coli, the specific preparation process of the fusion protein will be introduced below by taking Ib-AMP4-GFP and GFP-Ib-AMP4 as examples. Through the method of gene synthesis, synthesize the DNA expressing Ib-AMP4 and GFP protein, between two protein molecules (GGGSG) 4 In order to verify the difference in the membrane binding ability of...

Embodiment 2

[0025] Solid Phase Synthesis of Ib-AMP4-FITC

[0026] Antimicrobial peptides were synthesized by solid-phase synthesis using FMOC (9-fluorenylmethoxycarbonyl) as a protecting group. It was cleaved from the resin with 95% trifluoroacetic acid, 2.5% water and 2.5% triisopropylsilane (TIA). After repeated precipitation with diethyl ether, the polypeptide was purified by reverse-phase high-performance liquid chromatography. A C18 reverse-phase column was used in the purification: 0%-60% acetonitrile containing 0.05% trifluoroacetic acid was used as the mobile phase, and gradient elution was performed at a flow rate of 3 mL / min. The polypeptide was then dissolved in oxidation buffer (100 mmol / L ammonium acetate, pH 8.5) at a concentration of 1 mg / mL, and stirred continuously at room temperature for 3 days to fully oxidize and fold to form disulfide bonds. Finally, it was purified to over 95% by reverse-phase HPLC and freeze-dried for use. After amination of the C-terminus, comme...

Embodiment 3

[0028] Application of Ib-AMP4-GFP, thanatin-GFP and Ib-AMP4-FITC in clinical bacterial identification

[0029] Pick 10 clinical strains and inoculate them in fresh culture medium, cultivate them overnight, recover the bacteria by centrifugation and wash with 5% glucose solution for 3 times, then dilute the bacterial solution to a bacterial suspension with OD600=0.5. Take 1 mL of bacterial suspension and add 1 mL of different concentrations of Ib-AMP4-GFP, thanatin-GFP or Ib-AMP4-FITC, incubate in a shaker at 37 degrees Celsius for 20 minutes, wash the bacteria three times with PBS, and observe under a fluorescence microscope Fluorescence, and measure the green fluorescence intensity of the bacteria with a fluorescence spectrophotometer. The resulting data were used to classify bacteria using linear discriminant analysis. The results showed that after being treated with transmembrane protein, the bacteria were marked with green fluorescence, and the fluorescence intensity rang...

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Abstract

The invention relates to an organism identification method based on a membrane-bound complex. The membrane-bound complex at least consists of two parts including transmembrane protein and a signal molecule, and the transmembrane protein and the signal molecule are linked by a chemical bond or a flexible amino acid fragment. According to the provided rapid, simple and effective organism identification method, only an organism and the protein need to be mixed for incubation for 10 minutes, the fluorescence intensity of different organisms ranges from several thousands to several millions, the resolution among the organisms of different species is high, and the method has high repeatability and accuracy.

Description

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Claims

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Application Information

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Owner SOUTHEAST UNIV
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