Application of EGFP-CTA2-TAT fusion protein to preparation of fluorescent probe

An EGFP-CTA2-TAT and fusion protein technology, which is applied in the application field of EGFP-CTA2-TAT fusion protein in the preparation of fluorescent probes, and achieves the effects of improving sensitivity, no cytotoxicity, and good transmembrane activity.

Inactive Publication Date: 2018-03-06
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of using protein fusion technology to construct biomacromolecular proteins with good biocompatibility as fluorescent probes has not been reported yet.

Method used

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  • Application of EGFP-CTA2-TAT fusion protein to preparation of fluorescent probe
  • Application of EGFP-CTA2-TAT fusion protein to preparation of fluorescent probe
  • Application of EGFP-CTA2-TAT fusion protein to preparation of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 strain activation and glycerol tube preservation

[0042] (1) Plate preparation: LB solid medium ingredients, sterilized at 121°C for 30 minutes; light the alcohol lamp on the ultra-clean workbench with UV light on for 30 minutes in advance, and place the plate half-opened around the alcohol lamp; sterilized LB solid medium in the ultra-clean Cool the clean workbench to about 60°C, add ampicillin antibiotics (final concentration 50 μg / mL) and quickly pipette and shake well; finish pouring the plate before the medium solidifies, half-open the plate after pouring, open the workbench for ventilation, and cover it when the medium solidifies Wrap in plastic wrap for storage.

[0043] (2) Inoculation: a tube of 5mL LB liquid medium, first add 2.5μL ampicillin antibiotic stock solution (mother solution concentration 100mg / mL), then add 50μL E.coli BL21(DE3) containing recombinant plasmid pET22b-EGFP-CTA2-TAT Strain preservation solution; culture at 37°C, 200r / min...

Embodiment 2

[0047] The expansion culture of embodiment 2 target strains and the inducible expression of target protein

[0048] (1) The E.coli BL21 (DE3) monoclonal strain bacterial liquid containing the recombinant plasmid pET22b-EGFP-CTA2-TAT after the activation made in Example 1 is used in a volume ratio of 1:100 in a clean bench Inoculate into LB liquid medium containing ampicillin antibiotic (final concentration 50 μg / mL); then place at 37°C and culture overnight at 200 r / min on a shaker for 12 hours;

[0049] (2) On the ultra-clean workbench, transfer the bacterium liquid cultivated overnight in step (1) to the Erlenmeyer flask containing ampicillin antibiotic (final concentration 50 μg / mL) and LB liquid medium according to the volume ratio of 1:50; then Place on a 200r / min, 37°C shaker for 4 hours;

[0050] (3) Add IPTG with a final concentration of 1mmoL / L to the bacterial solution after the expanded culture in step (2); induce on a shaking table at 37°C and 200r / min for 5h;

...

Embodiment 3E

[0052] Example 3 Extraction and purification of EGFP-CTA2-TAT fusion protein

[0053] (1) Suspend the wet bacteria obtained in Example 2 in lysis buffer (PBS buffer) (wet bacteria: lysis buffer = 1 g: 10 mL); add lysozyme (final concentration 80 μg / mL) and use magnetic Stir evenly with a stirrer; put the above mixture in the refrigerator for repeated freezing and thawing, if the bacterial solution is still viscous after repeated freezing and thawing, add DNase for further cracking;

[0054] (2) When the bacterium in step (1) is fully lysed to ensure that it is no longer viscous, use a refrigerated centrifuge to centrifuge at 4°C and 10,000r / min for 10min, discard the precipitate and collect the supernatant to obtain a supernatant protein solution;

[0055] (3) Chromatographic column wet packing (Ni-Sepharose 6FF (His tag purification resin)), connect the chromatographic instruments (nucleic acid protein detector, peristaltic pump, chromatographic column) to take over each tube...

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Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to application of EGFP-CTA2-TAT fusion protein to preparation of a fluorescent probe. A TAT part contained in the structure of the EGFP-CTA2-TAT fusion protein is transmembrane peptide which can help a designed fluorescent probe to run through a cell membrane into the interior of a cell; a CTA2 part contains an endoplasmic reticulum positioning sequence KDEL which can achieve targeted positioning of an endoplasmic reticulum in the cell; the fusion protein can achieve real-time microscopic tracing detection of the endoplasmic reticulum in a living cell and the corresponding physiological change thereof ultimately by exciting EGFP to emit green fluorescence. Compared with an ordinary endoplasmicreticulum staining agent, the fusion protein has the characteristics of good biocompatibility, safety, non-toxicity and higher targeting performance and detection sensitivity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and particularly relates to the application of an EGFP-CTA2-TAT fusion protein in the preparation of fluorescent probes. Background technique [0002] In recent decades, with the establishment of some new molecular recognition mechanisms, the development and application of new materials, and the advancement of science and technology, more and more bioluminescent probes have been developed for basic research in life sciences. The objects of its research have transitioned from various ions or molecules in homogeneous solutions to ions, small molecules and biomacromolecules (enzymes, nucleic acids, proteins, etc.) in living organisms. In addition to constantly pursuing the construction method of bioluminescent probes with higher sensitivity and better selectivity, scientific researchers pay more attention to the application of bioluminescent probes in the in situ detection and real-time monit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N21/64
CPCC07K14/00C07K2319/01C07K2319/10C07K2319/60C12N15/62C12N15/70G01N21/6486
Inventor 王甜甜王华倩林丹敏何华锋
Owner GUANGDONG UNIV OF TECH
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