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Targeted anti-cancer polypeptide for inhibiting signal transmission of MKK7-JNK pathway and application of polypeptide

A technology of MKK7-JNK and signal transmission, which is applied in the field of medicine, can solve problems such as application limitations, and achieve the effect of enhancing curative effect

Active Publication Date: 2020-06-02
深圳市健翔生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Biological macromolecules play an important role in the treatment of many diseases, but due to the natural barrier function of the cell membrane, this makes some therapeutic value but no cell membrane penetration The application of molecules in the pharmaceutical field is greatly limited

Method used

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  • Targeted anti-cancer polypeptide for inhibiting signal transmission of MKK7-JNK pathway and application of polypeptide
  • Targeted anti-cancer polypeptide for inhibiting signal transmission of MKK7-JNK pathway and application of polypeptide
  • Targeted anti-cancer polypeptide for inhibiting signal transmission of MKK7-JNK pathway and application of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. In vitro binding experiment between TAT-RAMK-1 and MKK7

[0022] The coding sequence of SEQ ID NO.1 in the sequence listing was synthesized, and the coding sequence was ligated between BamHI and XhoI restriction recognition sites of pGEX4T-1. Expressed in Escherichia coli E. Coli BL21 (DE3) strain, and affinity purified to obtain the fusion protein GST-TAT-RAMK fused to GST and encoding the sequence. The in vitro binding activity of TAT-RAMK-1 and MKK7 kinase was detected by the Pull-Down method. 20 μg BSA, 10 μg his-MKK7 were respectively mixed with 5 μg of GST protein and GST fusion protein GST-TAT-RAMK in PBS, pH7.4 buffer Incubate with 30 μl Ni2+-NTAAgarose at 4°C for 4 hours, collect Ni2+-NTAAgarose by centrifugation and wash 3 times, discard the supernatant, and collect the remaining protein bound to Ni-Agarose plus SDS loading buffer. Protein samples were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue G250 to detect obvious bound ...

Embodiment 2

[0023] The solid-phase synthesis of embodiment 2.TAT-RAMK-1

[0024]Weigh the resin and put it into the polypeptide solid-phase reactor, add appropriate DMF to swell for more than half an hour. DMF was withdrawn, Fmoc deprotection reaction was carried out with de-resin protection agent, and placed on a shaking table for 10 min. Remove the protective solution, wash with DMF and DCM for 3 times, take a small amount of resin from the reactor into a test tube, wash with ethanol for 2 times, detect and record the color with the ninhydrin method, prepare for feeding, and enter the amino acid condensation reaction. According to the amino acid sequence SEQ ID NO.1 of TAT-RAMK-1, take the corresponding Fmoc amino acid and HOBt, dissolve them in DMF, add DIC under ice bath to activate for 5min, put them into the reactor, stir the reaction, after 1-2h, from Take a small amount of resin from the reactor in a test tube, wash it twice with ethanol, and detect it with the ninhydrin method. ...

Embodiment 3

[0027] Example 3. Effect of TAT-RAMK-1 on the MKK7-JNK pathway in liver cancer cells

[0028] Take the human liver cancer HepG2 cells in the logarithmic growth phase by 4×10 5 Cells / well were inoculated in a 24-well plate, cultured with fresh RPMI-1640 medium containing 10% FBS for 24 hours, and the cells were intervened with (5 μM, 10 μM, 20 μM) respectively, and a negative control group was set at the same time. After 24 hours of intervention, wash the cells with pre-cooled PBS solution, add 500 μL RIPA lysate, repeatedly pipette and mix, place on ice for 30 minutes, centrifuge at 4°C and 1200 rpm for 15 minutes, absorb the supernatant, and collect the total cell protein. Perform SDS-PAGE electrophoresis and transfer to PVDF membrane. Phosphorylated JNK antibody and phosphorylated MKK7 antibody were used to perform Western blot respectively to detect the phosphorylation of MKK7 and JNK in tumor cells after polypeptide intervention. The result is as figure 2 As shown, the...

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Abstract

The invention discloses a polypeptide for inhibiting signal transmission of an MKK7-JNK pathway in tumor cells and an application of the polypeptide. The polypeptide has an amino acid sequence shown in SEQ ID NO.1 in a sequence table. The polypeptide provided by the invention can enter cells, specifically bind to MKK7 in vivo and in vitro, further inhibit the signal transmission of the MKK7-JNK pathway by inhibiting the binding of the MKK7 to RACK1, and finally inhibit the growth of tumor cells.

Description

technical field [0001] The invention belongs to the technical field of medicines, and in particular relates to a polypeptide specifically inhibiting MKK7-JNK pathway signal transmission, and its application in the preparation of medicines for treating cancer diseases in which RACK1 is overexpressed and JNK is abnormally activated. Background technique [0002] JNK is one of the important members of the MAPK family. JNK acts on cytokines (such as tumor necrosis factor-α, interleukin-1, epidermal growth factor) and environmental stress (osmotic pressure, electrical damage, ultraviolet radiation, oxidative damage) through cascade activation. respond. Its upstream kinases include MKK4 and MKK7, which can phosphorylate the 183-threonine and 185-tyrosine of JNK at the same time, so that JNK can be double-phosphorylated and activated. In addition, MKK4 can also activate P38, while MKK7 only specifically activates JNK. After activation, JNK can participate in cell proliferation, c...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11A61K45/06A61K38/10A61P35/00
CPCC07K7/08A61K45/06A61K38/10A61P35/00
Inventor 支钦李新宇付玉清邱心敏
Owner 深圳市健翔生物制药有限公司
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