Compound polypeptide molecule for targeted killing of cancer cells and preparation method thereof
A cancer cell and molecular technology, which is applied in the field of compound polypeptide molecules that target and kill cancer cells and its preparation, can solve the problem that cracking peptides cannot target and kill cancer cells, achieve targeted killing of cancer cells, prevent damage, simple effects
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[0036] The preparation method of the composite polypeptide molecule provided by the embodiment of the present invention has simple process and low cost, and can be realized on a polypeptide solid-phase synthesizer. The finally obtained composite polypeptide molecule includes gallic acid, cleavage peptide, and links between gallic acid and cleavage peptide The MMP-2 specifically recognizes the degraded polypeptide substrate, which can achieve the effect of targeting and killing cancer cells.
[0037] In one embodiment, after synthesizing the complex polypeptide molecule, a step of separating and purifying by high performance liquid chromatography is also included. Specific separation process: Dissolve the peptide sheared from the resin, filter it with a 0.2 μm filter membrane, and separate it through a C18 reverse column. The mobile phase is water and acetonitrile, and the peptide peak is collected (monitored at an ultraviolet wavelength of 225 nm).
Embodiment 1
[0039] Example 1 Research on the effect of splitting peptide
[0040] The lytic peptide can destroy cancer cells and normal cells that are harmful to humans by lysing biofilms. This phenomenon can be verified by CCK-8 activity test.
[0041] (1) 5000-8000 human osteosarcoma cells MG-63 were seeded in a 96-well plate, cultured overnight in DMEM containing 10% FBS, and the culture conditions were 37°C, 5% CO 2 , and then use different concentrations (0, 5 μM, 10 μM, 20 μM) of the cleavage peptide (there are four kinds, respectively: SEQ.ID.No.2, SEQ.ID.No.3, SEQ.ID.No.3, SEQ.ID.No.3, SEQ. .ID.No.4, SEQ.ID.No.5) replace the culture medium, after continuing to culture for 24 hours, the cell survival rate is detected by CCK-8 reagent.
[0042] It was found that the above four lytic peptides can effectively kill half of the cancer cells at a concentration above 5 μM, indicating that the lytic peptides have good anticancer activity. The cleavage peptides SEQ.ID.No.3 and SEQ.ID.No.5...
Embodiment 2
[0045] Example 2 Research on the Molecular Action of Composite Polypeptides
[0046] 5000-8000 human kidney epithelial cells 293-T were seeded in 96-well plates and cultured overnight in DMEM containing 10% FBS at 37°C, 5% CO 2 , and then use the composite polypeptide molecules (wherein, MMP-2 specifically recognizes the degraded polypeptide substrate as SEQ.ID.No.1) of different concentrations (0, 5 μM, 10 μM, 20 μM) of DMEM dissolved in 10% FBS to be cleaved The peptide is SEQ.ID.No.5) to replace the medium, and after continuing to culture for 24 hours, the cell viability was detected by CCK-8 reagent.
[0047] The result is as Figure 5 As shown, since gallic acid neutralizes the positive charge of the cleavage peptide through the negative charge, it effectively inhibits the killing effect of the complex polypeptide molecule on normal cells.
[0048] Based on the above examples, it can be known that the examples of the present invention neutralize the positive charge of t...
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