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GPR1 antagonistic polypeptide and derivative and application thereof

A derivative, antagonistic technology used in biotechnology and biomedicine

Active Publication Date: 2019-06-28
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As GPR1 is one of the members of GPCRs, there is still no drug that directly targets GPR1 to treat clinical cancer.

Method used

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  • GPR1 antagonistic polypeptide and derivative and application thereof
  • GPR1 antagonistic polypeptide and derivative and application thereof
  • GPR1 antagonistic polypeptide and derivative and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Panning, amplification, purification, sequencing and synthesis of GPR1 antagonistic polypeptide LRH7-G6.

[0077] This example is mainly for the purpose of screening positive phages that specifically bind to GPR1, and then by amplifying and purifying the positive phages, extracting phage single-stranded DNA (ssDNA) for sequencing, analyzing and comparing the obtained sequences, and finally synthesizing high-purity phages. The antagonistic polypeptide LRH7-G6.

[0078] details as follows:

[0079] 1. Establishment of 293T cell line with permanent high expression of GPR1: 293T-GPR1 + / + / LRH

[0080] ①Select vigorously growing luminescent human 293T cells, and the day before transfection, use 5×10 5 cells / well, inoculated in a 6-well plate, cultured until the second day, the cell fusion degree was 60%;

[0081] ② Transfect on the second day, take one culture well of a 6-well plate as a unit, dilute 3 μg of plasmid with 200 μL of opti-MEM medium, and dilute 6...

Embodiment 2

[0099] Example 2 The GPR1 antagonistic polypeptide LRH7-G6 can effectively alleviate the inhibitory effect of chemerin on the cAMP signaling pathway.

[0100] (1) Cyclic adenosine monophosphate (cAMP) ELISA:

[0101] ① Cell plating: Wild-type 293T cells and 293T cells with high expression of GPR1 (293T GPR1 + / + ), with 5ⅹ10 5 Each well was inoculated in a 6-well cell culture plate, and the medium volume of each well was 1 mL. After being placed in an incubator and cultured for 24 hours, it was starved overnight, and LRH7-G6 polypeptides with different concentration gradients (3 μM, 0.3 μM, 0.03 μM) were added. , Fosklin (25μM) and chemerin (30nM) for 6h;

[0102] ②Sample preparation: Add 300 μL of cell lysate to each well, place at 4°C for 20 minutes, scrape and collect cells with a cell scraper, mix them upside down, centrifuge at 12,000 rpm for 10 minutes, and collect the supernatant;

[0103] ③ Determination of sample concentration: the sample concentration is determined b...

Embodiment 3

[0111] Example 3 GPR1 antagonistic polypeptide LRH7-G6 can effectively inhibit calcium (Ca 2+ ) inflow effect.

[0112] ① Cell plating: Wild-type 293T cells and 293T cells with high expression of GPR1 (293T GPR1 + / + ), with 5ⅹ10 3 Each cell / well was inoculated in a 96-well cell culture plate, the volume of medium in each well was 200 μL, placed in an incubator for 24 hours, and then starved overnight;

[0113] ②Reagent configuration: dissolve probenecid into 1mL buffer solution to prepare probenecid with a concentration of 250nM, shake well, add to fluorescent reagent for use;

[0114] ③Remove the cell culture medium, add LRH7-G6 polypeptide and chemerin (0.3nM) with different concentration gradients (30 μM, 3 μM, 0.3 μM, 0.03 μM, 0.003 μM) for 30 minutes, and then add 100 μL of the above fluorescent reagent to each well;

[0115] ④ Place at 37°C for 30 minutes, then at room temperature for 30 minutes;

[0116] ⑤Measure the fluorescence absorbance at excitation light 494nm...

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Abstract

The invention discloses a GPR1 antagonistic polypeptide and a derivative and application thereof and particularly relates to a GPR1 antagonistic polypeptide shown in SEQ ID No.1-4 and a derivative thereof. The derivative of the binding peptide is a product obtained in the mode that conventional modification is conducted on a GPR1 binding peptide amino acid side chain group and an amino terminal ora carboxyl terminal of a GPR1 antagonistic polypeptide fragment or a product obtained in the mode that a label for detection or purification of polypeptide or protein is linked to the GPR1 antagonistic polypeptide. The binding peptide and the derivative thereof can be combined with GPR1 in vitro, by stopping combination of chemerin and GPR1, increase of the cAMP concentration is promoted, the (Ca2+) influx caused by chemerin is inhibited, effective micromolecule treatment drugs are provided for diseases, such as breast cancer, of high-expression GPR1 receptors, and the GPR1 antagonistic polypeptide can be widely applied in the fields of medicine and biology.

Description

technical field [0001] The present invention relates to the field of biotechnology and biomedicine, specifically, the present invention is the female reproductive disease target GPR1 receptor antagonistic polypeptide LRH7-G6 and its derivatives and applications. Background technique [0002] Breast cancer is the second deadliest cancer in the world after lung cancer, and it continues to destroy the life and health of millions of women and their families around the world. According to the latest statistics from the International Cancer Research Center of the World Health Organization, there were 1.67 million new cases of female breast cancer worldwide in 2012, accounting for 22.9% of all female malignant tumors; 460,000 women died of breast cancer, accounting for 13.7% of all female malignant tumor deaths %, accounting for 1.7% of all female deaths, approximately 1 in 4 cancer-related women develop breast cancer. The age-standardized incidence rate of female breast cancer in...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11A61K38/09A61P35/00A61P1/16A61P3/10A61P29/00A61P15/00
Inventor 张键代小勇肖仲琳张保珍赵华山陈指龙韩金雨
Owner SHENZHEN INST OF ADVANCED TECH
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