A novel tumor targeting therapy polypeptide and its use

A tumor-targeting, new technology, applied in the field of biomedicine, can solve DNA damage, attack DNA and other problems, achieve the effect of small side effects, reduce the concentration of use, and improve the therapeutic effect

Active Publication Date: 2022-03-01
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in clinical practice, most chemotherapy and radiotherapy drugs directly or indirectly attack DNA, causing DNA damage.

Method used

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  • A novel tumor targeting therapy polypeptide and its use
  • A novel tumor targeting therapy polypeptide and its use
  • A novel tumor targeting therapy polypeptide and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Ubiquitination modification of RAD51 and foci detection

[0071] 1.1 Primary MEF cell culture, see general method 1.

[0072] 1.2 In vivo ubiquitination experiment, see general method 2.

[0073] The results showed that compared with WT (MEF-Atg7+ / +), in KO (MEF-Atg7- / -), the ubiquitination band of RAD51 disappeared, and the ubiquitination of RAD51 was mediated by ATG7, see figure 1 a.

[0074] 1.3 Detection experiment of ubiquitin activating enzyme E1 activity.

[0075] 1.3.1 His-ATG7 protein purification.

[0076] 1.3.1.1 Cell transfection experiment.

[0077] HEK293 cells were transfected with his-ATG7 plasmid, and HEK293 was inoculated on a 10cm culture dish according to the general method. After lysing for 30 minutes, the lysate was centrifuged at 13000 rpm, 4 degrees for 20 minutes, and the supernatant was taken after separation.

[0078] 1.3.1.2 GE His-tag protein purification prepacked column to purify his-ATG7 protein.

[0079] The His-tagged p...

Embodiment 2

[0091] Example 2 Detection of foci in RAD51 nucleus

[0092] 1.1 The effect of ATG7 deletion on RAD51 nuclear foci.

[0093] General method 1 Culture the MEF cells of Atg7 knockout mice, and detect the foci formed in the nucleus of RAD51 by immunofluorescence according to general method 3.

[0094] The results showed that in KO (MEF-Atg7- / -) compared with WT (MEF-Atg7+ / +), RAD51 nuclear foci formation was significantly reduced, ATG7 mediated, RAD51 nuclear foci formation, see figure 2 A, figure 2 B is the statistical result.

[0095] 2.2 Verification of RAD51 ubiquitination modification sites.

[0096] 2.2.1 Position of RAD51 ubiquitination modification

[0097] Construct a Flag-RAD51-Myc tagged plasmid with a Flag at the N-terminal and a Myc tag at the C-terminal, use general method 4 to inoculate and transfect Hela cell lines, and general method 3 for immunofluorescence detection.

[0098] The results showed that Flag-tagged foci were detected in the nucleus, while My...

Embodiment 3

[0105] Embodiment 3 The effect of polypeptide Rap of the present invention adjuvant chemotherapeutic drugs

[0106] 3.1 Human endometrial cancer KLE cell line was cultured according to general method 4, inoculated on a 10cm culture dish with a density close to 80%, given Cisplatin (cisplatin) and Rap stimulation, and after 24 hours, according to general methods 2 and 3, the RAD51 expression was detected ubiquitination level.

[0107] The results show that Rap reduces the ubiquitination modification of RAD51, see image 3 a.

[0108] 3.2 The human endometrial cancer KLE cell line was cultured according to general method 4, stimulated with Cisplatin (cisplatin) and Rap, and after 24 hours, the DDR pathway was detected according to general method 3.

[0109] It was shown that Rap inhibits the DDR pathway, see image 3 b.

[0110] 3.3 The human endometrial cancer KLE cell line was cultured according to general method 4, stimulated with Cisplatin (cisplatin) and Rap, and after ...

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Abstract

The present invention provides a tumor-targeting polypeptide, including: (a) a novel tumor-targeting therapeutic polypeptide; (b) a fusion protein formed by the polypeptide (a) and a penetrating peptide element; (c) a fusion protein formed by the polypeptide (a) or ( b) A derivative polypeptide derived by adding, deleting, or changing one or more amino acids and having effects on RAD51 ubiquitination modification, foci formation in the nucleus, and reduced DNA repair function; (d) the amino acid sequence corresponds to C of the RAD51 protein end (331-339aa), or a fusion protein formed by the C-terminus and the penetrating peptide element, and the polypeptide or fusion protein (d) can affect the ubiquitination modification of RAD51, the formation of foci in the nucleus, and reduce the DNA repair function. By administering the polypeptide of the present invention, only the ubiquitination modification of RAD51 is affected, thereby affecting the formation of foci in the nucleus, and the side effects are small; the concentration of chemotherapy drugs can be effectively reduced, and the drug sensitivity of tumor cells can be improved.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a novel polypeptide with targeted therapeutic effect on tumor cells and its composition. Background technique [0002] Cancer is currently the number one killer threatening human health. According to the 2018 Global Cancer Annual Report, the incidence and mortality of cancer in China rank first in the world. In our country, there is one cancer patient in every 65 people, and more than 5 people die of cancer every minute. Drug therapy is the main means of cancer treatment, however, the drug resistance of tumor cells is one of the main reasons for the failure of current cancer treatment. Therefore, enhancing the drug sensitivity of tumors is a hot spot in the field of anti-tumor research. [0003] The mechanism of tumor drug resistance is a very complicated process. Current studies believe that the main molecular biological mechanisms of tumor drug resistance include enhanced...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K19/00A61K45/06A61K38/17A61K47/64A61P35/00A61K33/243
CPCC07K14/4702A61K45/06A61K33/243A61K47/64A61P35/00C07K2319/10A61K38/00A61K2300/00
Inventor 关奕曹流邓诚思
Owner 中国医科大学
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