TCS-cell penetrating peptide-tumor protease substrate peptide fusion protein, preparation method and uses thereof

A protease substrate and fusion protein technology, which is applied in the field of TCS-penetrating peptide-tumor protease substrate peptide fusion protein and its preparation, can solve the problems of inhomogeneity of modified products, instability of free sulfhydryl groups, oxidation of sulfhydryl groups, etc.

Active Publication Date: 2018-08-28
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of these two modification methods are obvious
The former, due to the large number of lysine residues in the protein, will lead to inhomogeneity of the modified product, increasing the difficulty and cost of separation and purification of the modifie

Method used

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  • TCS-cell penetrating peptide-tumor protease substrate peptide fusion protein, preparation method and uses thereof
  • TCS-cell penetrating peptide-tumor protease substrate peptide fusion protein, preparation method and uses thereof
  • TCS-cell penetrating peptide-tumor protease substrate peptide fusion protein, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0079] Preparation of Recombinant Trichosanthin TCS

[0080] Prokaryotic Expression and Purification of Recombinant Trichosanthin TCS

[0081] a: The TCS recombinant expression plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain a strain containing the recombinant plasmid.

[0082] b: Culture the strain containing the recombinant plasmid in LB medium containing 100 μg / ml Amp in a constant temperature shaker at 37°C at 250 rpm until the logarithmic growth phase (absorbance value at 600 nm is 0.6-0.8), and add IPTG with a final concentration of 1 mM , expressed overnight (14h) at 25°C, 150rpm.

[0083] c: Collect the bacterial cells by centrifuging at 6,000 rpm at 4°C for 20 minutes.

[0084] d: Resuspend the bacteria in HEPES buffer (containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5‰ Tween 20, pH 8.5).

[0085] e: Use a probe sonicator with a power of 400 W to sonicate the cells for 30 min.

[0086] f: centrifuge at 12,000 rpm at 4°C for 20 m...

preparation Embodiment 2

[0092] Synthesis of TCS-penetrating peptide-MMP-2 substrate peptide fusion protein-lactoferrin linker

[0093] (1) Expression and purification of TCS-penetrating peptide-MMP-2 substrate peptide fusion protein

[0094] a: The recombinant expression plasmid of the TCS-penetrating peptide-MMP-2 substrate peptide fusion protein was transformed into Escherichia coli BL21 (DE3) competent cells to obtain a strain containing the recombinant plasmid.

[0095] b: Culture the strain containing the recombinant plasmid in LB medium containing 100 μg / mL Amp in a constant temperature shaker at 37°C at 250 rpm to the logarithmic growth phase (absorbance at 600 nm is 0.6-0.8), and add IPTG at a final concentration of 1 mM , expressed overnight (14h) at 25°C, 150rpm.

[0096] c: Collect the bacterial cells by centrifuging at 6,000 rpm at 4°C for 20 minutes.

[0097] d: Resuspend the bacteria in HEPES buffer (containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5‰ Tween 20, pH 8.5).

[0098] e: ...

experiment Embodiment 1

[0109] Determination of MMP-2 enzyme content in C6 and GL261 cells and their medium

[0110] The MMP-2 enzyme content in C6 and GL261 cells and their culture medium was detected by conventional Western Blot in the field. The specific method is as follows: HT1080 and HUVEC cells in the logarithmic growth phase were digested with trypsin and diluted to 1.5×10 5 The cell suspension of each cell / ml was transferred to a 6-well cell culture plate (Nunc Company), and 2ml of cell suspension was added to each well, and cultured for 4h with DMEM complete medium containing 10% calf serum (37°C, 5% CO 2 ). After the cells adhered to the wall, the medium was discarded, the cells were washed twice with PBS, and replaced with serum-free DMEM medium, 1ml per well, and continued to culture for 24h. The medium supernatant was collected, and the cells were lysed with cell lysate for 30 min, centrifuged at 4°C for 15 min, the supernatant was collected, the protein concentration was determined ...

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Abstract

The invention relates to a TCS-cell penetrating peptide-tumor protease substrate peptide fusion protein and a preparation method thereof, further provides a TCS-cell penetrating peptide-tumor proteasesubstrate peptide fusion protein-lactoferrin linker and a preparation method thereof, and further relates to uses of the linker in preparation of drugs for treatment of tumors. According to the present invention, the TCS-cell penetrating peptide-tumor protease substrate peptide fusion protein can increase the cytotoxicity of Trichosanthin on tumor cells, and is used for the treatment of malignanttumors; and the linker can penetrate through the blood-brain barrier and can be accumulated in brain tumors, and can responsively and selectively kill tumor cells with high matrix metalloproteinase-2expression due to the high matrix metalloproteinase-2 expression in tumor cells so as to inhibit the growth of tumors and prolong survival period.

Description

technical field [0001] The invention belongs to the field of biomedicine, more specifically, the invention relates to a TCS-penetrating peptide-tumor protease substrate peptide fusion protein and a preparation method thereof, and also relates to a TCS-penetrating peptide-tumor protease substrate peptide fusion protein -Lactoferrin linker and its preparation method and use. The linkers of trichosanthin (TCS) and penetrating peptide, tumor protease substrate peptide and tumor targeting ligand can be better enriched in malignant tumor sites, and can be activated by tumor protease highly expressed by tumor cells, and can be activated by penetrating Membrane peptides mediate transcytosis for tumor targeting. technical background [0002] Malignant tumors are one of the greatest threats to human life. According to the report of the World Health Organization, the incidence of cancer is increasing year by year, and it is showing a younger trend. Among all malignant tumors, brain ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C07K1/107C07K1/18A61K38/16A61P35/00
CPCA61K38/168C07K7/06C07K14/415C07K2319/10C12N15/70
Inventor 黄永焯陈应之
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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