Novel tumor targeted therapy polypeptide and application thereof

A new tumor-targeting technology, applied in the field of biomedicine, can solve the problems of DNA damage and DNA attack, and achieve the effects of less side effects, lower concentration, and improved drug sensitivity.

Active Publication Date: 2020-03-06
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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  • Novel tumor targeted therapy polypeptide and application thereof
  • Novel tumor targeted therapy polypeptide and application thereof
  • Novel tumor targeted therapy polypeptide and application thereof

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 invention provides a tumor targeted polypeptide, which comprises: (a) a novel tumor targeted therapy polypeptide; (b) a fusion protein formed by the polypeptide (a) and a cell-penetrating peptideelement; (c) a derived polypeptide which is derived from the polypeptide (a) or (b) by adding, deleting or changing one or more amino acids and has the functions of influencing RAD51 ubiquitination modification and intracellular foci formation and reducing DNA repair; and (d) a polypeptide of which the amino acid sequence corresponds to the C terminal (331-339aa) of the RAD51 protein, or a fusionprotein formed by the C terminal and a cell-penetrating peptide element, wherein the polypeptide or the fusion protein (d) has the functions of influencing RAD51 ubiquitination modification and intracellular foci formation and reducing DNA repair. By giving the polypeptide, only the ubiquitination modification of RAD51 is influenced, and the formation of intracellular foci is influenced, so that the side effect is small; and the use concentration of chemotherapeutic drugs can be effectively reduced, and the drug sensitivity of tumor cells is 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 Applications(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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